Sir Michael Wilshaw, head of Ofsted, said his inspectors found one independent religious school using dividing screens across the room to segregate men and women.
"I am writing again to report that Her Majesty's Inspectors (HMI) continue to find that staff are being segregated because of their gender in Muslim independent schools," Wilshaw writes in his letter sent yesterday to UK education secretary Nicky Morgan.
It adds that officials who inspected Rabia Girls' and Boys' School in Luton, east England, expressed their concern after the school insisted on "segregating men and women through the use of a dividing screen across the middle of the room" at the initial meeting.
"Male staff sit in one room and the session is simultaneously broadcast to female staff in another part of the school," the letter adds.
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The inspection was carried out earlier this month following the Department for Education's (DfE) request for Ofsted to carry out an emergency follow-up inspectionof the school already assessed as "inadequate".
Ofsted was so concerned about the behaviour in the school that they told the owner that the "school would remain in the inadequate category despite improvements being made elsewhere".
The private school was set up in 1996 to provide an Islamic education for young Muslims in Luton.
"It is completely unacceptable for women to be treated less favourably than men, and the advice note we have received from Ofsted on Rabia Girls' and Boys' School is concerning.
"We have referred this case to the EHRC (Equality and Human Rights Commission) so they can consider whether the school has breached the Equalities Act, and we will consider carefully the inspection report on the school to determine what action to take against any potential breaches in the independent school standards," said a DfE spokesperson.