The group B streptococcus or agalactiae streptococcus is gram positive bacteria. These organisms are normal flora of women genital and gastrointestinal tracts, which may cause early onset infection in new born infants and pregnant women. Severe infection of GBS may cause infection like meningitis, sepsis etc in new born infants. Determining the level of GBS carriage in the genital tract of pregnant mothers is useful for prophylaxis purposes and reducing the GBS infection. The present study was conducted on pregnant women, with pregnancy age of 35 weeks or more, who referred to Mustafa Khomeini, Ilam, from January 2014 to December 2015. Total 90 vaginal fluid samples were extracted from the vagina of pregnant women and Group B strep strains were identified by using cultivation methods. Antibiotic sensitivity of the samples were determined by disc-diffusion method. Data were analyzed using SPSS software, version 18. Chi-square and T-test were used as appropriated. Results of the present study revealed the incidence rate of Group B strep strains turned out to be 4.4% in pregnant women referring to Mustafa Khomeini, Ilam, during the study period. The Group B Strep colonization was also significantly associated (P<0.05) with the number of previous pregnancies. High prevalence of group B Streptococcus in pregnant women and their impact on the incidence of infection in both mothers and infants, screening is of crucial importance for pregnant women prior to giving birth. Considering high sensitivity of strains to Clindamycin, Vancomycin, and Cefazolin, it is recommended to consider these factors as the preferred treatment of infections caused by GBS.