Aim: To conduct epidemiological and ecological surveillance of cholera in freshwater environments. Methods and Results: A freshwater region of India was surveyed between April 2007 and December 2008. Vibrio cholerae was isolated from 59.5% of water and plankton samples (n = 357) and 35.5% of stool samples (n = 290). Isolation from water was dependent on air (r = 0 44) and water temperatures (r = 0 49) (P < 0 01) but was independent of rainfall (r = 0 15), chlorophyll a (r = 0 18), salinity (r = 0 2) or pH (r = 0 2) (P > 0 05). Isolation from plankton was dependent on temperature of air (r = 0 45), water temperature (r = 0 44), chlorophyll a concentration (r = 0 42), pH (r = 0 23) and salinity (r = 0 39) (P < 0 01). Cholera cases correlated with rainfall (r = 0 82, P < 0 01) and chlorophyll a concentration (r = 0 42, P < 0 05), but not with air temperature (r = 0 3, P = 0 37). Vibrio cholerae O1 possessed ctxB, ctxA, rstR and tcpA (ElTor), toxR, toxT, rtxA, rtxC, mshA and hylA. Among non-O1-non-O139, the distribution of virulence-associated and regulatory protein genes was heterogeneous with -0.7, 2 2, 94 77, 97 76, 99 25, 100 and 100% isolates being positive for tcpA, toxT, rtxA, rtxC, hylA, toxR and mshA, respectively. Two-thirds of non-O1-non-O139 isolates exhibited antibiotic resistance to various antibiotics that did not correlate with geographical site or time of origin for the isolates. RAPD and AFLP showed V. cholerae to be a diverse bacterium. AFLP demonstrated separate lineages for non-O1-non-O139 and O1 isolates. Conclusion: Environmental parameters played a significant role in the emergence and spread of cholera and the abundance of V. cholerae. But based on virulence gene profiling and genetic fingerprinting, the possibility of origin of toxigenic isolates from nontoxigenic environmental isolates seems unlikely in freshwater environs of India. Significance and Impact of the Study: This study explains the ecology, epidemiology and seasonality of cholera in freshwater environs.