To explain the sudden appearance and rapid spread of cholera in Latin America in January 1991, molecular techniques were used to define Vibrio cholerae 01 isolates from around the world. Restriction fragment length polymorphisms of rRNA and ctxA genes,DNA sequence of cholera toxin B subunit gene ctxB, and multilocus enzyme electrophoresis data were used to characterize 197 isolates. Worldwide, there are at least four distinct toxigenicEl Tor V. cholerae 01 clones: the seventh pandemic (Eastern Hemisphere), US Gulf Coast, Australian, and Latin American. Nontoxigenic V. cholerae O1 previously isolated in Brazil, Mexico, and Peru are unlike current toxigenic isolates. The Latin American clone probably represents an extension of the seventh pandemic into the Western Hemisphere, while the US Gulf Coast clone most likely evolved separately. These data willbe useful in monitoring the spread of cholera, determining the origin of outbreaks in both hemispheres, and implicating specific vehicles of transmission.