Representational difference analysis (RDA) is a recently developed technique used for amplifying genetic differences between two closely related genomes. We compared RDA and a modified version of RDA to examine genomic differences between the two Vibrio cholerae serogroups that cause epidemic cholera, O1 and O139, and between the two biotypes of the 01 serogroup. With both techniques, we recovered several sequences known to be found only in V. cholerae O139 but absent in its presumed progenitor, V. cholerae 01 El Tor. A greater number of unique fragments were generated in comparing the two V. cholerae 01 biotypes, consistent with the probable greater genetic differences between the two biotypes.