While urbanization in a sub-Saharan African (SSA) context can lead to greater independence in women, various sociological, biological, and geographical factors in urban areas may keep women at a higher risk for HIV than men. Access to major roads during Malawi's transition into rapid urbanization may leave women disproportionately vulnerable to HIV infection. It is not well established whether women who report to health clinics closer to major roads have higher or lower levels of HIV. In this study we explored the spatial heterogeneity of HIV prevalence among pregnant females in Lilongwe District, Malawi. Using Geographic Information Systems, we visually represented patterns of HIV prevalence in relation to primary roads. HIV prevalence data for 2015 were obtained from 44 antenatal clinics (ANC) in Lilongwe District. ANC prevalence data were aggregated to the administrative area and mapped. Euclidean distance between clinics and two primary roads that run through Lilongwe District were measured. A correlation was run to assess the relationship between area-level ANC HIV prevalence and clinic distance to the nearest primary road. ANC HIV prevalence ranged from 0% to 10.3%. Clinic to major road distance ranged from 0.1 to 35 km. Correlation results ( r= -0.622, p = 0.002) revealed a significant negative relationship between clinic distance to primary road and HIV prevalence, indicating that the farther the clinics stood from primary roads, the lower the reported antenatal HIV prevalence. Overall, the clinic catchments through which the major roads run reported higher ANC HIV prevalence. Antenatal HIV prevalence decreases as ANC distance from primary roads increases in Lilongwe, Malawi. As urbanization continues to grow in this region, road distance may serve as a good indicator of HIV burden and help to guide targeted prevention and treatment efforts.