Uropathogenic E.coli (UPEC) is the source of numerous toxins that cause nosocomial and community-acquired UTIs, including -hemolysin, a secreted lipoprotein that is a powerful and ubiquitous cytolysin that belongs to the (RTX) family. Between March 2021 to December 2021, 123urine samples were collected from patients with urinary tract infections who were admitted Al-Hilla General Teaching Hospital in Al-Hilla City, from both sexes (male and female). Out of (123), 56(45.5%) were positive culture for E. coli which identified by selective media, biochemical tests, Vitek 2 system and 16s RNA gene specific primer by the presence of (1492 bp) compared with allelic ladder, while other causes recorded 67(54.5%). DNA purification Kit supplemented by the manufacturing company Geneaid, (Korea). Primers were used and PCR condition to amplify sequences of the 16SrRNA and hlyA genes. Amplification products were separated by electrophoreses. The gene sequencing for 16SrRNA and HlyA gene was done by sanger sequencing method and phylogenic analysis. The study pointed to the number of mutation that happen with HlyA gene, as α-hemolysin gene varies greatly between local and international strains.