The investigation into the scrapping of the West Coast Main Line contract must be carried out by an independent expert, Labour has urged.
The decision to award the franchise to FirstGroup was scrapped over "technical flaws" in the bidding process.
The government has since announced a review of what went wrong, but Labour says this should not be carried out by a Department of Transport (DfT) member.
The DfT insisted its review is independent.
The West Coast route serves 31 million passengers travelling between London, the West Midlands, the north-west of England, North Wales and the central belt of Scotland.
FirstGroup had initially beaten current operator Virgin Trains to win the 13-year franchise.
But the award was halted after Virgin took legal action over the decision not to renew its contract. The tender will now be rerun.
Transport Secretary Patrick McLoughlin is expected to explain the U-turn at the House of Commons on Monday.
His review of the "unacceptable mistakes" by officials during the tendering process will be carried out by Sam Laidlaw, who is the chief executive of Centrica and a non-executive director of the DfT.
Shadow transport secretary Maria Eagle said it was not appropriate that the investigation will be carried by a DfT non-executive director.
In a letter to the Transport Secretary, she accused ministers of pre-judging the outcome of the inquiry.
Ms Eagle wrote: "Considering the need to look thoroughly at the role of everyone involved in this process, including ministers and senior officials, it is difficult to see how anyone will have confidence in the conclusions reached by fellow members of the departmental board.
"This is not in any way to criticise or pass judgment on those appointed by you to carry out this work, but to ensure the conclusions can be accepted without criticism of the 'inside' nature of the inquiry."
She also criticised what she called ministerial attempts to "scapegoat" officials.
A DfT spokesman said: "The independent review into the Intercity West Coast franchise competition will be led by one of the country's most prominent business figures.
"Sam Laidlaw is the lead non-executive on procurement across government and is the person best placed to establish what went wrong rigorously and promptly.
"Mr Laidlaw is using independent external advisers and has been asked to provide an initial report by the end of this month."
A second independent review - on how to take forward the wider rail franchising programme - will be undertaken by the chairman of Eurostar, Richard Brown.