Clifford the Big Red Dog is the latest kids TV programme to introduce LGBT characters.
The reboot of the animated children's series features a character called Samantha with two mums, Dr and Ms Mulberry.
The lesbian mothers made their debut in an episode called "The Big Red Tomato/Dogbot", which aired on US network PBS Kids last month.
The introduction of the characters has been criticised by campaign group One Million Moms, which accused PBS of pushing an LGBT agenda.
"Discussion of such controversial topics and lifestyle choices should be left up to parents," it said.
"PBS KIDS should not introduce the LGBTQ lifestyle to young children. PBS KIDS should stick to entertaining and providing family friendly programming, instead of pushing an agenda."
It follows the recent announcement from PBS that Broadway star Billy Porter will be making a guest appearance on Sesame Street.
Porter is openly gay and regularly turns heads with his gender-bending tuxedo gowns on the red carpet.
Last year, PBS came under fire over a gay wedding on another children's favourite, Arthur.
One Million Moms said: "Many families have already discovered that PBS KIDS, largely supported by federal funds, is anything but family friendly."
It added: "Please take action and help get the word out about this ridiculous use of taxpayers' dollars to indoctrinate children to the LGBTQ lifestyle."
