Vibrio cholerae remains a major public health threat worldwide, causing millions of cholera cases each year. Although much is known about the evolution and pathogenicity of the O1/O139 serogroups of V. cholerae, information is lacking on the molecular epidemiology of non-O1/O139 strains isolated from patients who have diarrheal illnesses. We performed whole-genome sequence analysis and in vivo infections to investigate characteristics of V. cholerae O141 isolated from sporadic diarrheal cases in 4 countries. The strains formed a distinct phylogenetic clade distinguishable from other serogroups and a unique multilocus sequence type 42, but interstrain variation suggests that O141 isolates are not clonal. These isolates encode virulence factors including cholera toxin and the toxin-coregulated pilus, as well as a type 3 secretion system. They had widely variable capacities for intestinal colonization in the infant mouse model. We propose that O141 isolates comprise a distinct clade of V. cholerae non-O1/O139, and their continued surveillance is warranted.