An unusual strain of Vibrio cholerae O1 biotype El Tor harbouring multiple tandem copies of classical CTX prophage caused a cholera epidemic in Mozambique in 2004. However, the location of the classical CTX prophage in the genome of the Mozambique strain was unknown. In this study, pulsed field gel electrophoresis (PFGE) of the whole genome along with Southern hybridization experiments indicated that the classical CTX prophage present in the Mozambique strain is located in the small chromosome. To determine the CTX prophage integration site in the small chromosome of Mozambique strain, the 5'and 3' junctions of the prophage and small chromosome were PCR amplified, cloned and sequenced. Sequence analysis indicated that the prophage was integrated in the conserved dif site of the replication terminus region of the Mozambique strain. While using an O1 El Tor isolate VC44 as a control strain, which carries tandem copies of CTX prophage in its small chromosome like the Mozambique strain, it was unexpectedly detected that the strain VC44 also possesses classical cholera toxin B gene allele. Since the strain VC44 was isolated in India in the year 1992, it appears that the Mozambique strain has probably originated from a VC44-like strain.