Background: Urinary tract infection (UTI) is among the most common bacterial infections and possess significant healthcare burden. Escherichia coli is the most common cause of UTI accounting for about 70%% and a variable contribution from Proteus mirabilis, Pseudomonas aeruginosa and Klebsiella pneumoniae Patients are often treated as soon as bacteria are shown to be present by microbiological culture. Objective: To assess THE PCR results among culture positive and culture negative specimens of suspected UTI patients. Methods: This study was carried out in the department of Microbiology, Mymensingh Medical College during the period from July 2016 to June 2017. Urine specimens were collected and isolationand identification of major uropathogens (Escherichia coli Klehsiella pneumonias, Proteusmirabilis, and Pseudomonas aeruginosa) were done by standard microbiological procedure a biochemical tests. PCR was performed by using standard protocol with species specific primer for detection of fimH gene for Escherichia coli, fimK gene for Klehsiella paeunomiae, UreCfor Proteus mirabilis, ETA for Pseudomonas aeruginasa. Results: Out of 250 urine specimens, 200 specimens were isolated and identified by culture and different biochemical methods which were supported by microscopical examination and at the same timePCR could detect species specific genes in 201 specimens directly from urine of suspected UTIpatient Escherichia coli was responsible as a leading causative pathogen in both outpatient department and in patient department with a higher prevalence of 71.8% for outpatient department. On the other hand Pseudomonas aeruginosa Profeus mirabilis and Klebsiella pneumonia were more prevalent in in-patient department and it was 21.1%, 5.6% and 5.5% respectively, Among the 50 culture negative urine specimens, 14 (28%) showed PCR positive for Escherichia coli, Klehsiella pneumonia and Pseudomonas aeruginosa. Conclusion: This study revealed that, the prevalence of UTI is high n MMCH Single pathogen base uniplex PCR was found superior than standard culture and less time consuming. Because uniplex PCR could detect many (28%) culture negative cases.