The Edinburgh to Perth service, which leaves daily at 4.34pm, has just two carriages, and runs at a whopping 136% of planned capacity.
The two-carriage service carries 234 passengers but only has capacity for 172 both seated and standing.
The Scottish Liberal Democrats put in a Freedom of Information Request to Transport Scotland asking for a list of the ten most overcrowded services in Scotland.
Another Fife service featured in fourth place, as the Edinburgh to Glenrothes With Thornton service via Dunfermline runs at 117% capacity.
Both services have just two carriages each and the busiest portion of the journey is at Inverkeithing.
Scottish Liberal Democrat transport spokesperson Mike Rumbles MSP said: “Commuters and tourists are shelling out more of their hard earned money than ever before to perch in corridors on overcrowded trains.
“Rail fares have consistently risen faster than wage increases.
“We will never tempt motorists out of their cars and onto public transport if being packed in like sardines is what they have to look forward to.
“Extra capacity is desperately needed but we know that the new trains which are supposed to provide this won’t now be delivered in full until April 2019.
“This is well behind the original schedule.
“Not only are the new trains not in place, existing trains are busier than ever after some leases were allowed to expire.
“People want to see evidence that their ticket costs are being invested in improving services and infrastructure, but promises have yet to be delivered.
“It is time Scotland has a transport system fit for the 21st century.”
A Transport Scotland spokeswoman said: “We wholly appreciate the impact of overcrowding on rail services and, as such, actively publish the top ten busiest trains in an open and transparent fashion on an annual basis.
“Our £475 million investment in the new Hitachi trains, as well as £54 million in a fleet of fully refurbished High Speed Trains, will transform travel across our key routes and inter-city rail travel between our seven cities.
“We’ve extended the leases of more than 30 trains to support the on-going fleet introductions, providing more seats and improved on-board passenger facilities across many routes.
“By 2019 we will have increased capacity by 50% over 2007 levels and passengers across the country will reap the benefits of this. All this is helping to build the best railway Scotland has ever had.”
A ScotRail spokesperson said: “The work we are doing to introduce brand new and upgraded trains will significantly boost the number of seats available to our customers across the country.
“Customers across the network are already benefitting from the considerable investment in our trains and we are working round the clock to deliver even more.”
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