In an interview on "The Tonight Show with Jay Leno," Obama said he has "no patience for countries that try to treat gays and lesbians and transgendered persons in ways that intimidate them or are harmful to them."
"I've been very clear when it comes to universal rights, people's basic freedoms," Obama said.
Obama's comments come after Russian President Vladimir Putin signed new laws earlier this year that prohibit expressing support for gay rights or same-sex marriage.
The laws sparked controversy with many calling for a boycott of the Winter Olympics in Sochi, Russia, which will be held next year.
Relations between the United States and Russia have recently grown tense, largely over Edward Snowden, the government former contractor who leaked details on secret National Security Agency surveillance programs. Russian officials granted Snowden temporary asylum despite administration demands that he be returned to the U.S. where he faces espionage charges.
Obama said that recent tensions between the two nations would not impact the upcoming Winter Olympics.
"I think Putin and Russia have a big stake in making sure the Olympics work," Obama said. "I think they understand that for most of the countries that participate in the Olympics, we wouldn't tolerate gays and lesbians being treated differently."