Cholera has re-emerged as a major infectious disease in the recent past, with a global increase in its incidence. This study was focused on Prevalence of ace, tcpA, zot and ctxA genes in Vibrio cholerae strains associated cholera epidemic in 2005, Iran. Number of Thirty-nine strains of Vibrio cholerae related to cholera epidemic obtained from different provinces in 2005 in Iran. All the isolates identified by using standard bacteriological methods. Multiplex Polymerase Chain Reaction performed for identification of ctxa,tcpa,ace,zot. All the isolates were inaba  O1, Of thirty nine inaba 100% were positive for ace and zot,89.74% presented ctxa and 84.61% showed tcpA. It was found that zot and ace genes in all strains, ctxA genes was found in 89/74 per cent and tcpA genes in 84.6 per cent of strains. It has been suggested that the control of cholera epidemics is too big a task for a national diarrheal diseases control programmed. Although this may be true in the case of extensive epidemics, it should not be forgotten that large outbreaks of this kind are often the result of initial delays in detection and containment. A properly organized national control programmed provides the framework for quick detection and prompt containment and is the best means of ensuring preparedness for cholera control.