The Vibrio pathogenicity island (VPI) encodes the toxin-coregulated pilus and other virulence factors for Vibrio cholerae to colonize the human intestine to cause cholera. We assessed the level of genetic variation of VPI in nine nonpandemic isolates, and compared them with the sixth and seventh pandemic strains by sequencing c. 5 kb each from the start, middle and end regions of the VPI. Variation is similar among the three regions at around 2%, except for the tcpA gene, which has a much higher level of variation (23%). Numerous recombination segments were identified with sizes up to 2177 bp. Nearly all VPI genes sequenced have a ratio of synonymous to nonsynonymous substitutions considerably lower than that for housekeeping genes, suggesting that VPI genes are under positive selection pressure for change. The tagA gene was deleted or damaged in six isolates, which is likely to affect the efficiency of colonization of the human intestine. Two genes, orf2 and acfD, previously found to be translated differently in the sixth and seventh pandemic strains, were determined to be mutant in the seventh and sixth pandemic strains, respectively. These findings enhance our understanding of variation in the VPI, and of the pathogenic potential of VPI-positive environmental isolates.