Rolling strikes spanning 24 to 48 hours could hit every regional train line within weeks, if up to 600 drivers, station staff and train controllers vote to down tools.

The Rail, Tram and Bus Union (RTBU) lodged an application to take protected industrial action at the Fair Work Commission on Tuesday afternoon, which must be approved by a majority of members for the strike to go ahead.

The union is considering first targeting the Geelong and Warrnambool lines, followed by Ballarat, Maryborough, Bendigo, Seymour, Shepparton and Gippsland.

Commuters on many of these lines already face chronic overcrowding and unreliable services, and have endured many months of bus replacement services, due to the state government's works on major transport projects.

The union also plans to have one-hour work bans, which would lead to delays and cancellations, incurring fines for V/Line.

V/Line workers are calling for a six per cent annual pay rise over three years, while the state government-owned rail operator has put two per cent on the table. V/Line's enterprise bargaining agreement expired in June this year.

Secretary of the union's locomotive division Marc Marotta warned of a "long and bitter struggle", accusing V/Line of pursuing a "scorched earth policy towards employees".

"We've had something in the order of 15 or 16 meetings and we are getting nowhere," he said.

"We are being told rubbish in the meetings."

The union said that V/Line was removing driver training provisions, an eight-hour shift guarantee, public holiday loadings and other penalties in documents lodged with the Fair Work Commission.

"They are looking to strip away nearly every condition that workers have got at V/Line," he said.

A V/Line spokesman said any offer would need to be consistent with government wages policy.

"We'll continue to negotiate in good faith with our workers' representatives about the new V/Line operations workplace agreement," he said.

The Age