The move follows a decision to award additional payments to prison officers but not to other staff.
The majority of officers are men and the Public and Commercial Services union (PCS) said that not extending the payments amounted to discrimination.
The PCS said "Our women members' jobs have been rated independently as equal, so pay should be equally rewarded."
Prison officers will receive an extra £1,000 this year and another £1,000 next year on top of annual pay awards.
The payments were sanctioned by senior Scottish government ministers, including finance secretary Derek Mackay and justice secretary Michael Matheson.
The move will cost the prison service £3.7m in each year.
Similar payments were made in 2015 at a cost of £7.4m.
The women who are claiming discrimination work for the prison service in areas including administration, psychological services, HR, payroll, IT and procurement.
PCS national officer for Scotland, Lynn Henderson, said "These payments have been made despite there being no change in the role of the prison officers.
"It is scandalous that the Scottish government has knowingly sanctioned discriminatory treatment of hard-working women workers in our prisons.
"It flies in the face of their own Fair Work Principles which include the need for equality."
Almost 100 female workers have filed sex discrimination and equal pay claims against the Scottish Prison Service.