Castillo, an office manager and model, will be America's candidate in the Miss Universe pageant in June in Panama City, Panama.
Michelle Arnette, Miss Alabama, was first runner-up and home state favorite Nicole O'Brian of Texas was second runner-up.
The 51 contestants were trimmed down in advance to 10 semifinalists for Monday's evening gown and swimsuit competitions, but the results were not made public until the national broadcast on NBC.
Television personalities Daisy Fuentes and Billy Bush, who co-hosted the event, dedicated the pageant to America's military forces fighting in Iraq.
The five finalists were asked questions written earlier by their fellow finalists.
O'Brian's question was whether celebrities should use their status to speak out against the Iraq war.
"I don't think it's good for them to go against the president and the war," she replied. "Have faith in our country, have faith in our president and have faith in our freedom."
This was the first year that the Miss USA was seen on NBC after the end of a long association with CBS. The pageant is now co-owned by NBC and Donald J. Trump, who was in San Antonio for the ceremony.
The winner's package includes extensive travel opportunities, a $20,000 wardrobe, a soap opera appearance and $45,000 scholarship to the School for Film and Television in New York.
Judges included model Melania Knauss, soap-opera star Kristian Alfonso, pro football player Warren Sapp, actor Mekhi Phifer and former astronaut John Blaha.
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