In the present study, a total of 35 stool samples were collected in the Central Health Laboratory of Babylon Province from patients presenting invasive cholera disease. The period of collection was from November 2017 to December 2018. Identification of Vibrio cholerae species was carried out by conventional methods, biochemical tests, diagnostic kits and then confirmed by PCR-based assay targeting the ompW gene. The results of this study reported that O1 serogroups were the predominant serogroup among all clinical samples with a high rate of 94.3% (N = 33), while only two isolates of non-O1/non-O139 (NAG) (5.7%) were documented as a causative agent to cholera or cholera-like disease. The phylogenetic relationship among all 35 studied strains elucidated by using polymerase chain reaction (PCR)-based fingerprinting assay (ISSR-PCR). The results of this assay showed grouping of Inaba strains into different clusters indicating that these strains were genetically diverse. Furthermore, V. cholerae El Tor O1 Ogawa strain (OG1) was closely related to strains of Inaba serotype. In contrast, NAG strains (NAG1 and NAG2) were not genetically similar to any of Inaba or Ogawa strains indicating different clone origin.