Background: Group B Streptococcus (GBS) is a significant public health issue and a leading cause of chorioamionitis, puerperal endometritis and infectious morbidity in the newborn. The study objective was to determine the prevalence of Group B Streptococcus (GBS) colonization among HIV positive pregnant women compared with HIV negative pregnant women and to identify risk factors associated with GBS colonization among the study groups. Methodology: This descriptive cross sectional comparative study was conducted between July and December 2011, on 200 pregnant women attending antenatal clinic at OAUTHC who were divided into two groups: HIV positive group (n=67) and a control group consisting of HIV negative pregnant women (n=133), to assess the regional colonization by Group B Streptococcus (GBS). Vaginal and anal swabs were collected at 35-37 weeks of gestation and cultured in Todd-Hewitt broth, followed by a confirmatory test. For a control group with an anticipation proportion based on literature research of 14.1% and alpha=0.05, a sample size of 133 would have a power of 80% to detect a difference of 15% or greater with a study group in the proportion of 2:1. Patient sociodemographic characteristics and CD4 counts were extracted from the records and statistical analysis was done with SPSS 16.