OBJECTIVE: To investigate whether specimens obtained from the perianal area have a Group B streptococcal culture detection rate similar to anorectal specimens. METHODS: This is a prospective cohort study at a tertiary care university-affiliated teaching hospital. A total of 136 pregnant women between 33 and 40 weeks’ gestation were recruited. Three samples for Group B streptococcal culture detection were obtained from each subject in the following order: perianal sample, vaginoperianal sample, and an anorectal sample. The women were asked to rank their pain or discomfort with obtaining the anorectal sample. The vaginoperianal specimen is the standard sample obtained from antepartum patients in this clinic, and, therefore, it serves as the control. RESULTS: Of the 136 subjects, 26.5% of the control, vaginoperianal samples were positive for Group B streptococcal culture. In comparison, 27.2% of the anorectal specimens and 28.7% of the perianal specimens were positive for Group B streptococcal culture. There was no statistically significant difference in the detection of Group B streptococcal culture among the three sample sites. Evaluation of the pain experienced with an anorectal sampling showed that 68% of subjects ranked their pain between mild to moderate, and 5% noted severe pain. CONCLUSION: The Group B streptococcal detection rate was not different among the three sampling sites. Therefore, pregnant women do not need to be subjected to the additional pain of anorectal sampling to detect Group B Streptococcus.