The Telegraph has learned that the operator has asked the Serious Fraud Office to investigate.
The Transport Secretary, Grant Shapps, said: “There is clear, compelling and serious evidence that [Southeastern railway] has breached the trust that is absolutely fundamental to the success of our railways. When trust is broken, we will act decisively.”
Whitehall sources said that the Department for Transport investigation into the matter is ongoing and could yet uncover more undeclared sums.
The issue is understood to relate to “variable track access charges". The operator runs services on high-speed tracks, known as HS1, which is owned by a group of private investors.
Under its agreement, the Government would pay Southeastern a fee, which it was supposed to pay to HS1. Any excess ought to have been returned to Westminster, which in this case did not occur.
However, errors dating back to 2014 have been identified. Industry sources said that problems were noted during negotiations between the Government and Southeastern to agree a new national rail contract.
The two companies that ran Southeastern in a joint venture, FTSE 250 bus and rail operator Go-Ahead and state-backed French firm Keolis, said they had returned the money and were conducting a review of how the mistakes happened.
The two companies also run Britain’s biggest rail network, Govia Thameslink. Whitehall sources said that if investigations linked further discrepancies back to the owning groups, further sanctions could be taken.
Sources close to the operator insisted that the issue was an isolated incident and had not been replicated on the owners’ other line.
Go-Ahead's finance chief, Elodie Brian, who was previously Southeastern’s finance head, has stepped down with immediate effect.
David Brown quit as chief executive of Go-Ahead in May, but there is no suggestion that this was in relation to Tuesday’s revelations.