The number of breaches of rules on mixed-sex NHS wards rose by 98 last month, according to new figures.
Data from the Department of Health showed there were 559 breaches in England last month, compared to 461 in March.
The overall number of breaches has fallen by 95 per cent since data was first published in December 2010.
Health minister Simon Burns said the NHS has made “great strides” in tackling the issue.
He added: “The small rise in these figures is due to a handful of trusts. It shows why the NHS cannot be complacent about this, if we’re to continue the great progress made so far.
“Nobody should have to suffer the indignity of mixed-sex accommodation.
“Every unjustified breach is one too many and I urge those hospitals that have still to tackle this problem to look at the improvements in other hospitals and follow them. If they don’t, they will face fines of £250 for every breach.”
The Department of Health said three strategic health authorities - London, the North West and the South East coast - together account for 70 per cent of the breaches.
Of the 163 health trusts that submitted data, 123 (75 per cent) reported zero breaches.
Single-sex accommodation means patients of the same sex sharing sleeping, bathroom and toilet facilities, either in single rooms or single-sex wards.
Under the national guidance, men and women must have separate bays or rooms if sharing a ward. Intensive care and A&E are exempt from the policy.