Aslef has warned that modifications must be made to ScotRail's new Class 385 electric trains - or its drivers will refuse to work them.
The train drivers' union is concerned that the curved windscreen is causing reflections of other signals at night.
Drivers identified the problem on a recent evening test run between Glasgow Central and Paisley Gilmour Street.
The units are due to come into service on the Edinburgh Waverley to Glasgow Queen Street service in March.
Drivers reported that they could "view two or three signals when we should have been viewing only one".
Kevin Lindsay from Aslef, said: "ScotRail is trying to get Hitachi to come up with a solution but, so far, I'm afraid, they have failed.
"I've informed ScotRail that we won't allow these trains to come into service like this."
'Fit for purpose'
The union has called on Transport Minister Humza Yousaf to intervene.
Mr Lindsay said: "Aslef welcomes investment in new rolling stock and new infrastructure, but it has to be fit for purpose.
"That's why I'm calling on the minister to ensure these trains are safe when they come into service."
A spokesperson for Transport Scotland said: "The whole purpose of testing is to flush out potential issues.
"Drivers' input into that process is vital, having had that feedback it's imperative Hitachi and ScotRail work towards a solution.
"This means the Class 385s will only be brought into passenger service once this has been achieved and these trains are approved by the independent Office of Rail and Road (ORR)."
ScotRail has ordered 46 three-car and 24 four-car sets from rolling stock manufacturers Hitachi.
The trains are part of the Edinburgh Glasgow Improvement Programme (EGIP).
Initially, they will only run between the two cities, but there are future plans to introduce them on suburban routes south of Glasgow and to Dunblane, Stirling and Alloa.
A spokesperson for ScotRail Alliance said: "We test lots of elements of the train before it enters passenger service.
"The windscreen is just one element of our rigorous testing regime ahead of the introduction of our brand new trains."