The population structure of V. cholerae in Chesapeake Bay, United States, was analyzed and a simple procedure that employed only two biochemical tests in an abbreviated identification scheme, i.e., arginine dihydrolase and esculin hydrolysis, was developed. After enrichment in alkaline peptone water and selective plating on thiosulfate-citrate-bile salts-sucrose agar, all of the sucrose fermenting colonies identified by the two tests were confirmed as V. cholerae by the polymerase chain reaction. A non-redundant collection of 224 V. cholerae strains collected from 1998 through 2000 from Chesapeake Bay was analyzed for phenotype, genotype, and genomic fingerprint. A long-range enterobacterial repetitive intergenic consensus (ERIC) PCR method that was developed for this study provided fingerprint patterns that proved useful in assessing relatedness among the strains. Cluster analysis was done using three different methods and revealed three well separated, primary clusters: Cluster A, consisting of the majority of the isolates, including the toxigenic type strain for the species and luminescent strains of V. cholerae; a smaller Cluster B, with the noteworthy characteristic of low toxR gene homology; and Cluster M, consisting exclusively of V. mimicus. Another primary cluster, Cluster C, was also identified as a single clone of a sucrose-negative, luminescent, toxR-negative strain. Because V. mimicus formed a separate cluster with similar distance values as demonstrated by V. cholerae Cluster B, using both ERIC fingerprinting and DNA-DNA hybridization, and had phenotypic and genotypic traits and 16S rDNA sequences similar to V. cholerae, it is concluded that all of the primary clusters observed in this study, including V. mimicus, belong to a single species, V. cholerae. V. mimicus was judged to be the highest risk group of the non-toxigenic isolates, in terms of susceptibility to CTX-phi and possession of the heat-stable enterotoxin gene (stn). Approximately 11% of the V. cholerae strains that lacked the toxin-coregulated pili (TCP) and 50% of the V. mimicus strains were found to be susceptible to CTX-phi. In addition to the V. cholerae strains in the toxigenic subcluster, luminescent V. cholerae strains represented the next highest risk, since 14% of the luminescent strains were susceptible to CTX-phi and 33% were stn positive.