A multiplex PCR was developed to detect pre-CTXΦ and CTXΦ in Vibrio cholerae. A total of 115 V. cholerae were tested, of which 42 V. cholerae O1 and 18 V. cholerae O139 contained CTXΦ. Six V. cholerae O139 contained only pre-CTXΦ and three V. cholerae O1 and 23 V. cholerae O139 contained both pre-CTXΦ and CTXΦ. None of the V. cholerae non-O1 and non-O139 that were tested had pre-CTXΦ or CTXΦ. Results of Restriction Fragment Length Polymorphism (RFLP) analysis revealed the V. cholerae isolates possessed single or multiple copies of pre-CTXΦ and CTXΦ, always proceeded by a tandemly arranged RS1 element. Comparative nucleotide sequence analyses of the core region genes, orfU and zot, of 15 V. cholerae showed pre-CTXETΦ and CTXETΦ lineage with V. cholerae El Tor and pre-CTXClassΦ, pre-CTXCalcΦ, and CTXCalcΦ with classical V. cholerae O1 and O139. Two distinct types of ctxB were detected in V. cholerae O139. Multi-locus Sequence Typing (MLST) of seven V. cholerae housekeeping genes indicated clonal origin, irrespective of the presence of pre-CTXΦ and/or CTXΦ.