The debut was greeted with dismay by members of the Campaign for the Defence of the Traditional Cathedral Choir, who suspect that, although the girls will sing separately at first, they will end up merging with the boys.
Manchester Cathedral and St Mary's, in Edinburgh, already have mixed choirs.
Peter Giles, a former lay clerk at Ely, Lichfield and Canterbury, and chairman of the campaign, said: "There are only 100 or so boys' choirs in the world - and Britain has got about 70 of them. These very fragile musical miracles will all go eventually. We can't see why this has to happen simply because of the current politically correct climate."
Salisbury Cathedral was the first to recruit girls, in 1990. Since then, nearly a third of cathedrals in Britain have opened up their choir stalls.
The world-renowned choirs such as those at St Paul's Cathedral, Westminster Abbey, King's College, Cambridge, Christ Church, Oxford, and Canterbury have not done so, however.
Winchester is the most high-profile choir to change. "There has been an established male choir there for more than 900 years. If it goes now, it will never come back," Mr Giles said.
David Hill, the organist and master of choristers at Winchester Cathedral, denied that the boys' choir would suffer in any way.
The girls' contribution would be "complementary but utterly independent", he said. "Think of the boys' and girls' choirs as wines. They are both lovely, but they are different. It's not as if one is a weak, awful apple juice and the other a good burgundy.
"Having said that, the girls are starting out today and they have only been going two months, so they are a young wine."
Campaign members argue that boys will stop singing in choirs if it comes to be seen as a "girlie" thing to do.
"Quite the reverse," said Mr Hill. "We will be able to attract more boys on the grounds that they will only have to sing two rather than three services on a Sunday, which will give them more time with their families."
The dean of the cathedral, the Very Rev Michael Dean, said yesterday the response to the all-female choir was still being gauged. "It is still a case of wait and see."
The 17 girls in the newly formed Winchester girls' choir are aged between 11 and 16. They attend the nearby Pilgrims' School, rehearse three times a week and, as of yesterday, sing in one service a week. The total cost of running the girls' choir is in the region of pounds 30,000.
Michael Howard, formerly organist and master of the Choristers at Ely Cathedral, and a member of the campaign, said that girls' and boys' voices were fundamentally different. "There is no such thing as a female counter tenor," he said. "Little girls not only have unfocused voices but they are incapable of producing the dynamic range from pianissimo to forte."
On this, Mr Hill agreed. "There is a dolce sweetness to the character of the girls' voices," he said. "The sound is less robust than the boys'.
"It's about the way boys and girls approach things. I have no problem with that. We are not trying to make the girls conform to some kind of model, which certain people believe exists. For me, today is a totally uncontroversial day. It's just like a new addition to the family," he added.