A rule preventing patients of different sexes from sharing the same ward was breached more than 1,000 times at United Lincolnshire Hospitals Trust over a six-month period, figures reveal.
The Patients Association said the violations are an "affront to patients' dignity", and added that it is concerned over an increase in breaches nationally.
Hospitals have been expected to eliminate mixed-sex wards – except in justified situations, such as in intensive care – since 2010.
But NHS England figures show that between October and March, United Lincolnshire Hospitals NHS Trust recorded 1,039 breaches of mixed-sex accommodation rules.
This was compared to no breaches in the five months between October 2019 and February 2020 – the latest comparable period.
Data was not recorded between March 2020 and September 2021 because of the impact of the coronavirus pandemic.
And in April 2021, a policy to fine trusts a blanket rate of £250 for each breach of the rules was also dropped by the NHS.
The single-sex rules apply to sleeping accommodation, which includes any area where patients are admitted on beds or trolleys even if they do not stay overnight.
Across England, 16,576 breaches were recorded between October and March – up from 12,947 between October 2019 and February 2020, and the highest number for the period since 2010-11.
Between October 2014 and March 2015, there were just 1,740 breaches nationally.
The Patients Association said it understands the challenges the NHS faced during the pandemic, but it must now restore services to pre-Covid levels.
Rachel Power, chief executive of the charity which campaigns on behalf of patients, said: "The figures for March are of great concern – mixed sex wards are an affront to patients’ dignity.
"No patient wants to receive intimate, personal care on a mixed sex ward, and it's the sort of stress that doesn't promote recovery."
She added that urgent Government investment in social care could help reduce the number of breaches by allowing more medically fit patients to be discharged.
In March, there were 1.9 breaches of sleeping accommodation for every 1,000 hospital stays across England.
This was the second highest breach rate for any month since 2010-11.
At United Lincolnshire Hospitals Trust, the breach rate in March was 0.4 per 1,000 – down from 22.9 in February.
Jessica Morris, a fellow at independent health think tank the Nuffield Trust, said: "These situations will be distressing for patients, but staff are left with little choice.
“Breaches for mixed-sex accommodation were unfortunately already common across NHS trusts before the pandemic and are a symptom of hospitals running constantly close to capacity.
"The impact of Covid has seen performance against many targets slip further out of reach."
An NHS spokesman said: “The reported breaches illustrate the ongoing pressures on NHS providers during the Covid-19 pandemic.
"NHS staff work hard to maintain our patients’ privacy, dignity and safety – and most trusts have eliminated breaches."