A total of 111 clinical and environmental O1, O139 and non-O1/O139 Vibrio cholerae strains isolated between 1978 and 2008 from different geographical areas were typed using a combination of methods: antibiotic susceptibility, biochemical test, serogroup, serotype, biotype, sequences containing variable numbers of tandem repeats (VNTRs) and virulence genes ctxA and tcpA amplification. As a result of the performed typing work, the strains were organized into four clusters: cluster A1 included clinical O1 Ogawa and O139 serogroup strains (ctxA+ and tcpA+); cluster A2 included clinical non-O1/O139 strains (ctxA− and tcpA−), as well as environmental O1 Inaba and non-O1/O139 strains (ctxA− and tcpA−/tcpA+); cluster B1 contained two clinical O1 strains and environmental non-O1/O139 strains (ctxA− and tcpA+/tcpA−); cluster B2 contained clinical O1 Inaba and Ogawa strains (ctxA+ and tcpA+). The results of this work illustrate the advantage of combining several typing methods to discriminate between clinical and environmental V. cholerae strains.