HIV RNA detection in the newborn is the main diagnostic tool for vertical transmission. Most infections are thought to occur peri- or post-natally, hence preventive antiviral therapy administered days before and during delivery. This study used cord blood for molecular diagnosis, examined viral load and HIV-1 subtypes as determinants of transmission, and compared molecular variability of maternal, cord blood, and post-natal quasispecies. Ninety-seven seropositive mother-cord blood paired plasmas from Ghana were tested for HIV RNA. Viral load was quantified and a subgroup of 45 random women samples was typed and subtyped. HIV-1 from infected pairs was cloned, sequenced, and analyzed phylogenetically. The prevalence of HIV infection in pregnant women was 3.3%. 13/97 cord blood samples (13.5%) contained HIV RNA. No correlation between either viral load at labor (range 10(3)-10(7)) or HIV-1 subtype and in utero transmission was found. In both transmitting and non-transmitting mothers, 56% of HIV-1 strains were CRF02_AG. In three pairs, maternal and cord blood quasispecies were closely related, suggesting late pregnancy or perinatal transmission, while in four pairs, genetic distances suggested transmission earlier during gestation. Maternal viral load and genotype did not correlate with HIV-1 pre-natal transmission. HIV infection during gestation appears relatively frequent.