Along with many poll experts and opinion polls, the bookies had predicted a victory for the Remain camp with almost 80-90 per cent certainty and made Remain the odds-on favourites.
As a result, the vast majority of gamblers bet on Remain as opposed to Britain's exit from the EU.
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As the polls closed on Thursday night, bookmakers were offering 4-1 odds on an Out vote.
"Those of us who do this for a living have to face up to some tough questions today. The truth is that bookies do not offer markets on political events to help people forecast the results. We do it to turn a profit - or at least not lose too much - and in that respect, this vote worked out very well for us," said Matthew Shaddick, of Ladbrokes, one of the UK's biggest bookies.
He admitted the predictive power of the bookies will "be heavily criticised, understandably".
One theory why the bookies were so wrong was that most political betting was done by better-off customers, who backed Remain and drove down the odds.
A record amount was gambled, with Betfair taking more than 60 million pounds by Thursday afternoon, the largest ever for a political event.
While bookies did not suffer any monetary loss, it does reflect a hit on the reputation of Britain's bookies who have historically been more accurate about the results than even some exit polls.
Winners included Eurosceptic UK Independence Party (UKIP) leader Nigel Farage, who had bet put 1,000 pounds on Brexit.
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