Sheffield City Region has missed out on millions of pounds of transport funding in the Budget after the Chancellor favoured areas with a metro mayor.
Philip Hammond announced half of a new £1.7bn Transforming Cities Fund would be shared by six areas with elected mayors.
The remainder would be ‘open to competition’ by other cities in England.
Sheffield region will be fighting it out with the rest of the country after a legal challenge pushed mayoral elections back to May 2018.
Meanwhile, full devolution in Sheffield City Region - set to bring £900m and new powers - is on hold after Barnsley and Doncaster backed out to pursue a One Yorkshire ambition - which the Government has repeatedly said they can never join.
Richard Wright, executive director of Sheffield Chamber of Commerce, said: “It’s a reminder of just how much our city region is being penalised by not pushing forward on devolution and not having a mayor.
“The figure of how much we may have got in terms of investment is unknown – but our lack of progress means we will certainly lack that ‘firepower’ in terms of spending.”
Richard Flint, chief executive of Sky Bet, which employs hundreds of people in Sheffield and Leeds, said it was time to press ahead with “deals that are achievable.”
He said: “It is time to press ahead one way or another with deals that are achievable and start making use of the money the government is putting behind metro mayors.”
Mr Hammond underlined the Government’s preference for mayors by announcing several other schemes including a second devolution deal in the West Midlands, a new devolution deal with North of the Tyne and £123 million for the Redcar steelworks site to support the new Tees Valley mayor, Ben Houchen.
Chris Hearld, KPMG’s north region chair, said Yorkshire risked becoming a “straggler in the race for economic growth.”