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To investigate the molecular basis for serotype variation in Vibrio cholerae O1 and the genetic relatedness amongst different serotypes isolated from 2004 to 2008 in Iran.Despite the presence of all three serotypes of V. cholerae O1 (Ogawa, Inaba and Hikojima) in Iran in the last decade, the Inaba strains have been the dominated serotype. Sequence analysis of wbeT determined only a single substitution of G for A at position 295 in all Inaba strains resulting in a replacement of serine to proline. No difference was found in the copy numbers and profile of IS1004 between the classical and El Tor V. cholerae O1 strains, supporting the clonality amongst the isolates obtained over 5 years in Iran. In addition, Southern blots of HpaII-digested chromosomal DNAs of our Ogawa and Inaba isolates showed the presence of an incomplete copy of IS1004 for all isolates.IS1004 profiling can be a reliable method for analysis of clonal dissemination of V. cholerae. The results indicated that specific point mutation at a particular position within the wbeT of V. cholerae O1 strains in Iran may occur which, in turn, may result in serotype switching.Understanding the molecular basis for serotype conversion of V. cholerae and their genetic relatedness could give insights for the incoming cholera epidemic prediction and control.