The move will mean travellers having to self-isolate for ten days at home after returning, as well as taking two Covid tests.
Travel agents claimed Scotland had been “black-listed” while Edinburgh Airport said the country had “returned to the same lottery as last year”.
Transport Secretary Michael Matheson said: “Portugal is a popular destination for Scottish holiday makers and this change will clearly cause disruption to people’s travel plans.
“However, we have been forthright from the outset that the traffic light system is designed to protect the safety of the people of Scotland and the continued progress we are making as we come out of lockdown.
“Using the stringent methodology of the Joint Biosecurity Centre, it is clear the risk this new variant poses is now too great to allow unrestricted travel to Portugal, an approach that has been agreed by all four nations.
“International travel for holidaying purposes remains risky and subject to sudden change.
"We have said before people should think very carefully about travelling – and this latest development serves to underpin that advice – especially so given the prevalence and unpredictable nature of variants of concern.”
Seven countries will be to the red list at the same time, where ten days in a quarantine hotel on return is required: Afghanistan, Bahrain, Costa Rica, Egypt, Sri Lanka, Sudan, and Trinidad and Tobago.
The moves comes weeks after flights to Portugal from four Scottish airports resumed after First Minister Nicola Sturgeon said travellers would have to take Covid tests but not self isolate.
Easyjet is currently operating between Edinburgh and Lisbon, and Glasgow and Faro.
Ryanair is flying to Faro, Lisbon and Porto from Edinburgh, and to Faro from Aberdeen and Prestwick.
British Airways is flying between Edinburgh and Faro.
Jet2 has delayed the start of its Portuguese flights to July, while TUI has yet to start its flights.
Easyjet chief executive Johan Lundgren said: “This shock decision to add Portugal to the amber list is a huge blow to those who are currently in Portugal and those who have booked to be reunited with loved ones, or take a well-deserved break this summer.
"With Portuguese rates similar to those in the UK, it simply isn’t justified by the science.
“And to add no more countries to the green list when most of Europe’s infection rates are on a downward trend and many places are with low infection rates below that of the UK, such as the Balearics with 33 in 100,000 and Malta with just 12 in 100,000, makes no sense.
"[This is] especially when domestic travel is allowed within the UK, despite a number of cities having infection rates 20 times greater than much of Europe.”
"Yet the [UK] Government has torn up its own rule book and ignored the science, throwing peoples’ plans into chaos, with virtually no notice or alternative options for travel from the UK.
"This decision essentially cuts the UK off from the rest of the world.”
Aviation data firm Cirium said 119 flights were scheduled to depart from Scotland to Portugal this month, accounting for nearly 22,000 seats.
It said most – 76 – were from Edinburgh, with 17 from Glasgow and 13 each from Aberdeen and Prestwick.
The lion’s share of the flights are to Faro – 83 – with 21 to Lisbon and 13 to Porto.
A spokesman for Edinburgh Airport said: "We are disappointed for passengers who have planned trips to visit family and friends or to take a well-earned break and we encourage them to speak to their airlines to find out what happens next.
"These decisions create confusion and erode confidence in travel when we know people want and need to fly
"Public health is crucial to any recovery, but unfortunately we have returned to the same lottery as last year rather than using the time to develop a well thought out plan to allow the safe restart of travel.
"We cannot forget these decisions impact thousands of jobs and the livelihoods of people across Scotland, so it is important we find a system that is robust and reliable."
The Scottish Passenger Agents’ Association, which represents travel agents, said it represented a “systematic dismantling of Scotland’s travel sector”.
President Joanne Dooey said: “Travel agents have run out of words to describe their feelings of shock at the various measures which have been introduced - and revoked - since the start of the pandemic.
"We described the situation of ‘travel corridors’ last year as the hokey cokey as we didn’t know from one week to the next which countries would be in or out.
"This year, with the introduction of the traffic lights, all we have is a more colourful version of the hokey cokey – which is after all a comical and ridiculous child’s game.
“It’s a fallacy to say there is a green list for Scotland, as with the removal of Portugal, which was effectively the only country we could fly to from Scotland, we’ve added another colour – as Scotland seems black-listed.
“This isn’t just about those holidaying in the Algarve, who travel agents will have to work, for no income, to repatriate or those who will have to self-isolate for ten days and take expensive tests.
"It’s about the systematic dismantling of Scotland’s travel sector.”
Tim Alderslade, chief executive of Airlines UK, said: “This is a fresh body blow for families desperate to get away and see their loved ones this summer, and the hundreds of thousands of people who work in the travel industry, who are in grave danger of being forgotten.
“This is no way to treat passengers.”
A British Airways spokesperson said: “This is incredibly disappointing and confusing news, not just for aviation but also for our customers.”