An equal pay case is being taken against Network Rail (NR) on behalf of 34 women amid claims that female staff are earning thousands of pounds less than men for doing similar jobs.
The Transport Salaried Staffs' Association said it was taking the largest ever equal pay claim in the rail industry, claiming it could end up costing NR millions of pounds.
The union is due to outline details of the case at its annual conference in Cardiff, including claims for more than £25,000 in back pay for the 34 women, who work in middle management.
General secretary Manuel Cortes said the women were "the tip of the iceberg", adding that hundreds more claims could eventually be brought against the rail infrastructure firm.
He said an equal pay survey of 2,000 staff had revealed the pay gap of £4,500 at NR.
"That is just an average figure, in some cases the gap is as high as £10,000. This is completely unacceptable in a firm which receives the majority of its funding from the taxpayer.
"We are determined to end women being treated as second class citizens in the rail industry when it comes to pay. NR should set an example to the rest of the industry, bearing in mind the Equal Pay Act became the law of the land more than 35 years ago."
A Network Rail spokesman said: "Network Rail is committed to fair pay practices and has agreed to work with the TSSA to review our current systems."