It comes ahead of Flag Day on Sunday and as part of the celebrations marking 250 years of our nation's independence.
The ceremony was held at Veterans Memorial Auditorium.
The flag was retired at exactly 2:50 p.m. to signify two and a half centuries of our country.
The event was part of a nationwide effort to honor America's past - and look with hope towards its future.
A U-S military color guard presented the Stars and Stripes in front of dozens in Fresno Saturday to initiate the ceremony.
25 years ago, the now-retired flag was presented to Fresno's Veterans Memorial Museum by former Congressman George Radanovich.
This weekend, it was the centerpiece of an official flag retirement ceremony coordinated by the city of Fresno.
"This is the first flag retirement ceremony I've ever been at. So, it was something I wanted to experience. I wanted us to be the ones to host it in Fresno," said Fresno Mayor Jerry Dyer.
The event was part of the city's celebrations ahead of America's 250th birthday this July 4th and in honor of Flag Day on June 14th.
A band shared the stage with veterans and local and national leaders, who each delivered messages about the unifying force of the nation's flag.
"It represents one nation, under God, indivisible, with liberty and justice for all."
Messages of unity are a core principle of Fresno and America 250 celebrations in Central California and across the nation.
"The politics of division may be successful short-term, but they're not the American way. The American way is cherishing our fellow Americans," said Rep. Jim Costa.
In that spirit, Costa presented a new flag to the city and museum: a Betsy Ross flag featuring the 13 stars representing equality among the 13 colonies of young America surrounding the number 250.
The new flag will replace the flag retired on Saturday.
It's a symbol tying the country's past to its hope for the future.
"Be proud to be part of this country, not perfect, there is no perfect nation in the world. But it's the best nation in the world," said World War II veteran Norris Jernigan, the last remaining survivor of the crew aboard the Enola Gay, responsible for the atomic bombing of Hiroshima in 1945. "It's worth enlisting and taking part in protecting."