Urinary tract infection (UTI) is the most common disorder caused by bacterial agents in pregnancy. This study aimed to investigate the association of TLR4 gene polymorphisms with susceptibility to urinary tract infection caused by Gram positive bacteria in pregnant women a total of 39 clinically confirmed UTI pregnant women and 35 healthy control were enrolled in this study. Urine and blood samples were collected in Babylon teaching hospital for gynecology and pediatrics in Babylon province, Iraq during the period from January 2018 to February 2019. Urinary sample isolates were identified by traditional method. DNA was extracted using blood samples. TLR4 gene amplification and SNP genotyping was done using PCRRFLP. Cytokine profile was assessed using ELISA technique. The present study showed 22 (56.4%) isolates was Staphylococcus species and; 17 (43.6%) was streptococcus species. the results showed the presence of homozygous variant in 16 patient from the study group at site 299 (A/G). twentyhomozygous variant and 9 heterozygous variant at site 399 (C/T). Serum IL17 results showed a significant difference (p value= 0.016) between patients and controls. Serum TNF-α results showed a significant difference (p value= 0.021) between patients and controls.