He announced Network Rail had said a "safety-critical" component had failed, which would affect the planned start of electric trains in July.
The setback follows the original completion date being put back from last December because of other difficulties with the project.
The problem will delay overhead electric power cables going live at the end of this month.
That will have a knock-on effect on ScotRail drivers being trained to drive electric trains on Scotland's busiest route.
Industry sources told The Scotsman that the latest problem could also postpone the start of an all-electric service of brand new trains on the line from December.
It is understand the faulty equipment is part of the overhead power lines and will have to be replaced - possibly over the full length of the 46-mile route.
Mr Yousaf said any further delay after the earlier problems announced last summer would be "extremely disappointing".
The Â£742 million project will enable faster-accelerating, longer and greener electric trains to replace diesels on the route via Falkirk High.
Electric trains from other routes are planned to initially take over some services by the end of July.
They were due to be followed by the first of a fleet of brand-new Japanese Hitachi trains from September, with the last diesel trains replaced in December.
They will be progressively lengthened between December and the end of next year, when Edinburgh to Glasgow journeys are due to be cut by about ten minutes to 42 minutes.
Mr Yousaf said the delay further highlighted the need for the devolution of control over Network Rail so the Scottish Parliament could properly hold it to account over its projects and spending north of the Border.
The news came in a letter from Network Rail chief executive Mark Carne.
A Network Rail spokesman said: “There is an emerging issue around some of the installations on the Edinburgh-Glasgow electrification programme.
"We are working hard to assess the implications of this and won’t be commenting further until we have completed that work.”