Infection with E .coli represented the most highest causes of Urinary tract infections among community. Molecular techniques such as polymerase chain reaction (PCR) have become most important technique for more rapid and accurate for detection bacterial causal organisms in species level. E. coli based on detecting gene targets lac Z which encode for the enzyme ?-galactosidase is important target gene, this gene is responsible for energy production through break down of disaccharide lactose to the glucose and galactose. The urine samples were collected from UTI’s patients initially diagnosed according to the presence of pyuria, a classical culture methods for detection of E. coli was recorded the presence of E. coli in 20 out of 30 UTI’s samples (66%) while in molecular method by using polymerase chain reaction (PCR) for detection lac Z gene, E. coli was detect in 18 out of 30 UTI’s samples (60%), this difference between classical culture and PCR for identification of E. coli was non significant (p=0.05), and this may be due to the presence of evolved beta-galactosidase lac Y in some strain of E. coli which have the same galactose permease activity as lac Z galactosidase. So the present work strongly encouraged using PCR technique as a novel, perfect and fast test for E. coli diagnosis on molecular level. Further study for developed multiplex for both lac Z and lac Y galactosidase is highly recommended.