The trust has been landed with a fine of £1,750 for the breach of the rules.
The breach happened in November when a patient in the hospital's critical care unit was waiting to be moved to a ward but a bed was not available.
It was the first breach since June, when the trust was fined £750. Hospital bosses today said such a breach was rare.
Gwen Nuttall, chief operating officer at the Royal Wolverhampton NHS Trust, said: "The trust works very hard to ensure that mixed sex breaches are minimised and rarely has any of these incidents.
"Unfortunately, in this incident, it took longer than we would have liked to identify appropriate gender beds for patients being transferred from ITU onto a ward."
Information about the breach was contained in a report to a meeting of hospital bosses, which took place last month.
It said: "There was the first single sex breach in ICCU since June, with a patient requiring a ward bed, seven patients were involved."
Driving out mixed sex wards has been a long-held ambition of the past few Governments. In April 2011, a new system of fines was introduced to try to eradicate the issue.
Single sex accommodation means patients share sleeping, bathroom and toilet areas with only members of the same sex.
Hospitals can be fined £250 for each day a patient is kept in mixed sex accommodation.
In the past few years, the number of breaches of mixed sex accommodation rules has fallen dramatically.
In March last year, the Express & Star revealed hospitals in the Midlands were winning their war on mixed sex wards, with just 15 patients across the whole region forced to sleep in them in the month before.
Since 2011, hospitals have had to provide monthly reports on the number of times the rules are broken.