Environmental Vibrio cholerae strains belonging to the non-O1/non-O139 serogroups are natural inhabitants of freshwater including estuarine environments. Recent findings indicated that chironomids (Diptera: Chironomidae), the most widely distributed insects in freshwater, serve as a natural reservoir of these bacteria. Here we study the role of chironomids, particularly exuviae as carriers and as a monitoring tool for the distribution of V. cholerae in the environment. During a survey conducted in India (June 2006), 326 V. cholerae non-O1/non-O139 isolates were isolated from chironomid egg masses, larvae and exuviae. In addition, a heat-stable enterotoxin (nag-st) positive strain was isolated from exuviae during the local cholera outbreak. We identified 62 different strains in a subset of 102 isolates by analysis of variable number of tandem repeats (VNTR), demonstrating a high variation of V. cholerae on hosting chironomids. Our results show that chironomids can both maintain and distribute this overwhelming diversity of environmental V. cholerae strains, including toxigenic ones. Exuviae proved to be an efficient tool for the monitoring of environmental V. cholerae, offering simple, direct and practical access for on-shore collection. Finally, finding toxigenic V. cholerae on chironomids in endemic areas, together with molecular typing, may potentially improve monitoring of cholera in the future.