Muslim girls who wear the hijab to primary school will be asked why they wear it by inspectors.
The reasons given will then be recorded in school reports, amid concerns girls are being forced to wear the headscarf by their parents.
Amanda Spielman, the chief inspector of schools, announced the move on Sunday.
She said schools could be in breach of equality laws if they only require girls to wear religious garments.
There were also concerns wearing the hijab at such an age could be seen to be sexualising young girls, as they are traditionally worn by young women after puberty as a sign of modesty in the presence of men.
“In seeking to address these concerns, inspectors will talk to girls who wear such garments to ascertain why they do so in the school,” Ms Spielman said.
Responding to the move, Harun Khan secretary general of the Muslim Council of Britain, said: "It is deeply worrying that Ofsted has announced it will be specifically targeting and quizzing young Muslim girls who choose to wear the headscarf.
"It sends a clear message to all British women who adopt this that they are second class citizens, that while they are free to wear the headscarf, the establishment would prefer that they do not."
He added: "It is disappointing that this is becoming policy without even engaging with a diverse set of mainstream Muslim voices on the topic."
It comes after a survey for The Sunday Times found 18 per cent of 800 primary schools in England list the hijab as part of their uniform policy - most as an optional item.
Previous research by the National Secular Society (NSS) found 42 per cent of Islamic schools, including 27 primary schools, have a uniform policy requiring girls to wear a hijab.
The NSS wrote to Justine Greening, the education secretary, to ask for Muslim girls to be given "free choices," adding that forcing children to wear the hijab is "entirely at odds with this fundamental British value and with wider human rights norms on children’s rights".
The letter also expressed concern several non-Islamic schools were "acceding to fundamentalist pressure to incorporate the hijab into their uniform".