The bookies on Market Street and Queen Square shut down suddenly over the past week, with bosses saying the shops had become "uneconomic".
It follows the shock closure of KFC on Queen Square, with the high number of empty units prompting fears that Wolverhampton is turning into a "ghost town".
Over the summer William Hill announced plans to close 700 shops across the country, blaming the Government's decision to reduce the maximum stake on fixed-odds betting terminals (FOBTs) to £2.
A spokesman for William Hill said: "In July, William Hill announced that we had entered into a consultation process with retail colleagues over plans to close around 700 licensed betting offices.
"This follows the Government’s decision to reduce the maximum stake on B2 gaming products to £2 on 1 April 2019.
"Unfortunately these two shops in Wolverhampton are now uneconomic and have closed as a result of this.
"We are utilising redeployment and voluntary redundancy where possible and will provide full support to anyone impacted by redundancy.”
Wolverhampton has been hit by a string of store closures in recent months, including Beatties.
Starbucks, Footlocker, Patisserie Valerie, TH Baker have all closed stores on Dudley Street this year, while the BHS building remains empty while B&M and Wilko prepare to move in.
Conservative councillor Paul Singh said he had serious concerns about the future of retail in the city centre.
He said: "It is one thing when your anchor stores start to close down, but when the bookies and fast food restaurants are closing then it really is time to start worrying.
"It has been clear for the last 10-15 years that the city centre has needed to evolve, and it just hasn't happened.
"The controlling Labour group needs to improve conditions for traders. There has been so much disruption – including issues with car parking and access to the shopping areas – that it is hardly a surprise that people are going elsewhere.
"We are proud Wolverhampton people who want the best for our city, but at the moment it is at risk of turning into a ghost town."
Councillor Milkinder Jaspal, from the ruling Labour group, said the city centre was facing an "extremely challenging" period.
"I am really concerned about the number of store closures and would like to see rents reduced on units," he said.
"That is the only way we can hope to attract new businesses in these challenging times."