Restriction fragment length polymorphism analyses of the array of CTXΦ prophages in strains CRC262 and CRC266 of Vibrio cholerae O139 revealed the presence of copies of complete CTXΦ and pre-CTXΦ prophages coexisting at a single chromosomal locus in each strain. Restriction pattern and comparative nucleotide sequence analysis revealed pre-CTXΦ precursors of both the El Tor and Calcutta lineages. Thus, we hypothesize that two precursor variants independently acquired cholera toxin genes and gave rise to the current El Tor and Calcutta CTXΦ prophages. We discuss the implications of these results in terms of the evolution and origin of the current diversity of CTXΦ prophages.