Escherichia coli are gram-negative bacteria that cause urinary tract infections (UTIs). UTIs have affected a significant percentage of humans yearly due to bacterial infection. Our study aims to determine the prevalence of resistance genes in E. coli towards sulfamethoxazole. This study included (490) patients with UTIs, and the urine samples were cultured on media. The patients were admitted to the Medical City in Baghdad to treat UTIs. 116 E.coli isolates were isolated from urine specimens, 35 isolates of them were resistant to trimethoprim/sulfamethoxazole, and 81 isolates were sensitive to trimethoprim/sulfamethoxazole; the E. coli isolates were submitted to multiplex PCR to detection some resistance genes (Sul1, sul2) after detected the isolates by PCR depending on 16S rRNA. Our study showed that identified E. coli was (91-99%) depending on the number of the examined samples by the Vitek 2 system. The molecular study included extraction of chromosomal DNA from (53) E. coli isolates; 35 samples were taken resistant to antibiotics, while from the total of 81 sensitive isolates, only 18 sensitive samples were taken from that are the most sensitive to Timethprime/sulfamethoxazole, then identification by 16S rRNA gene. Detection of Sulfonamides resistance genes included sul1 and sul2. The results showed the 16S rRNA gene identification found in all E. coli isolates and the detection of antibiotic resistance genes. The resistant isolates with the Sul1 gene prevalence were 11(31%), while the sensitive isolates with Sul1gene were 1(6%). Moreover, the resisted isolates with Sul2 gene prevalence was 8(23%), while the sensitive isolates with the Sul1 gene were 0(0%). The numbers of the resistant isolates were (11) and (8) that carry the Sul1 gene and Sul2 gene, respectively, while the numbers of the sensitive isolates were (1) and (0), respectively. We can conclude that a high percentage of Sul1 gene and Sul2 genes in E. coil isolated from UTIs were high. Keywords. UTI, Sul1, Sul2, resistant gene, trimethoprim-sulfamethoxazole