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<p>Maternal rectovaginal colonization is the major risk factor for early-onset neonatal sepsis due to Group B (GBS), a major cause of early life morbidity and mortality. Transmission generally occurs perinatally from colonized mothers to infants. Vaccines targeting a subset of GBS serotypes are under development, but GBS epidemiology remains poorly understood in many African nations. We performed a cross-sectional study of GBS colonization among pregnant women at two sites in Botswana, a country with minimal prior GBS carriage data. We found a rectovaginal colonization rate of 19%, comparable with studies in other regions; however, we also noted a striking predominance of serotype V (> 45% of strains). Although further studies are required to delineate the burden of invasive GBS disease in Botswana and the generalizability of type V epidemiology, these data provide a useful baseline for understanding the potential local impact of GBS prevention strategies, including vaccines.</p>