Construction union UCATT is claiming that some subcontractors are not directly employing workers on a section of the line being overseen by a joint venture between Hochtief and Murphy.
It also alleges that one of the subcontractors is failing to provide regular rest breaks, in breach of health and safety rules.
The first phase of the CTRL was dogged by claims that the firms working on it were avoiding PAYE and National Insurance by making their workers pretend to be self-employed. The unions estimated that only one in four workers was directly employed.
As a result of the criticism, Rail Link Engineering, the consortium in overall control of the project, said in February that it would audit firms' working practices and dismiss those found to be questionable.
Bryan Rye, a UCATT regional officer, has now written to the Hochtief/Murphy joint venture in charge of section 320 of the scheme at Swanscombe, Kent, to complain that its subcontractors are still not employing workers on proper terms.
The letter, which is addressed to Leslie Forrest, Hochtief's managing director, has been seen by Building. It claims that a substantial number of operatives who should be directly employed have CIS self-employment documents.
In the letter Rye also says he has received complaints that workers with a named subcontractor are being denied rest entitlements.
UCATT general secretary George Brumwell said he would also write to Hochtief and Murphy to call for an investigation into working practices.
He said: "We have already taken up the issue with Bechtel [part of Rail Link Engineering] and we will now call for Hochtief and Murphy to investigate this particular site."
Forrest said those working on the scheme for Hochtief should have contracts of employment agreed with the unions.
He said: "Complaints that workers are not being properly employed will be carefully considered by the joint venture board."
A CTRL spokesperson said audits were taking place. She added: "It is our policy that all labour should be directly employed. We expect our main contractors to ensure that."