There is now no official timeline for when the trains will begin running, despite work on the new line - the first phase of East West Rail - being completed in December 2024.
A driver-only operation (DOO) - where the train driver is in control of the opening and closing of the doors - has been a contentious issue for rail unions in the past and led to strikes by members of the RMT at operators such as Southern and South Western Railway.
An RMT spokesman said: "On Chiltern and East West Rail, we can confirm that management have written to the union spelling out their plans for DOO.
"We are seeking talks and RMT's standing policy is that we are opposed to DOO."
A source at train drivers' union Aslef told the PA news agency it believed "there should be no extension" of driver-only operation on Britain's railways, as it was "clearly not safe" for some services.
A spokesperson for Chiltern Railways said: "We are working with the Department for Transport (DfT), trade unions and other industry partners to deliver the first stage of East West Rail for customers and businesses.
"As well as creating nearly 100 new permanent jobs at Chiltern, this new service will deliver immense benefits across the region, so we are eager to ensure that these benefits are realised for the community as soon as possible."
A report published by the National Audit Office in December 2023 said East West Rail represented a "£6-7bn investment".