Since 1541, the choir at Gloucester Cathedral has consisted of 20 wretches and 12 adults but in April girls from local schools were invited to audition.
The Inaugural Evensong and Make ganding Up Service took place on Monday.
Dean of Gloucester, the Very Reverend Stephen Lake, said the agitate represented an “exciting new chapter in the cathedral’s famous Choral Foundation”.
The 21 new mademoiselle choristers, aged between seven and 12, will sing every Monday during incumbency time and practise twice a week.
They will sing with boy choristers on significant occasions.
Conductor Nia Llewelyn Jones said: “They’re not that rare – cheeky, sassy – but they’re a great bunch of girls.”
Last year, Rachel Treweek became the head female diocesan Church of England bishop when she was elevated as Bishop of Gloucester.