While girls have been allowed to sing alongside boys for several years, this is the first time they've been allowed to sing alone in the choir since King Henry VIII created it in 1539.
A special cake was made to celebrate the event.
Gloucester Cathedral and the Mayor of Gloucester took to social media to commemorate the occasion.
@GlosCathedral wonderful time last night at the all girl choristers. History in the making. Girl power. pic.twitter.com/pKl1AAPWrJ
— Neil Hampson (@GlosMayor) November 8, 2016
Last night our Girl Choristers made history by singing their first ever service. What a debut! #evensong #smashedit pic.twitter.com/NKn3NbDWfw
— Gloucester Cathedral (@GlosCathedral) November 8, 2016
Canon Celia Thomson told the BBC: "I hope that lots of people will come to hear them and experience the worship with them singing."
Conductor Nia Llewelyn Jones added: "They're not that different to the boys.
"Cheeky, sassy, but they're a great bunch of girls."
