Earlier, Rick Santorum staked two primary wins in Mississippi and Alabama.
With 99 percent of the vote counted in the Aloha State, Romney picked up 3,407 votes to Santorum's 1,930 votes (25 percent). U.S. Rep. Ron Paul of Texas was third with 1,425 votes (19 percent) and former House speaker Newt Gingrich won 848 votes (11 percent).
Romney, a former Massachusetts governor, released a statement rather than make a speech after losing to Santorum and Gingrich in the Alabama and Mississippi GOP primaries.
Romney aides told The New York Times the candidate was now unlikely to reach the 1,144 delegates needed to secure the Republican presidential nomination for at least two more months.
"We will be increasing our delegate count in a very substantial way after [Tuesday's primaries]. We are even closer to the nomination," Romney's statement said after he got 29 percent of the vote in Alabama and 30 percent in Mississippi.
Romney also won the American Samoa Republican caucuses.
Aides said Romney made a statement rather than a traditional election-night speech because he was in transit.
Santorum, a former senator from Pennsylvania, won with 35 percent in Alabama and 33 percent in Mississippi. Gingrich, a former U.S. House speaker from Georgia, garnered 29 percent and 31 percent, respectively, and Paul, a Texas congressman, tallied 5 percent and 4 percent, respectively.
"We did it again," Santorum told supporters in Lafayette, La., a state where the GOP will hold a primary March 24.
He said he would campaign in Puerto Rico, Missouri and other states with upcoming primaries.
"We will compete everywhere," he said, calling on conservatives to "pull together."
In a swipe at Romney, Santorum added: "For someone who thinks this race is inevitable, he spent a whole lot of money trying to be inevitable. Who would have ever thought in the age of media that we have in this country today that ordinary folks can defy the odds day in and day out?"
Gingrich, who finished a close second in both states, noted he earned about as many delegates as his rivals and pledged to take his candidacy to the Republican National Convention in August.
"The elite media's efforts to convince the nation that Mitt Romney is inevitable just collapsed," Gingrich told a subdued crowd in Birmingham, Ala.
"If you're the front-runner and you keep coming in third, you're not much of a front-runner," he said.
He separately told WZZK-FM, Birmingham: "The biggest bloc is saying Romney, but it's not a big enough bloc to be a majority. We now are beginning to think he will literally not be able to get the delegates to get the nomination."
The Alabama and Mississippi primaries, along with the Hawaii and American Samoa caucuses, pushed the Republican presidential contest past the halfway mark.
Voters in 27 states have now weighed in on the process of selecting delegates for the party's nominating convention.
The race moves to the Missouri caucuses Saturday and the Illinois primary Tuesday.