We analyse the distribution of HIV-1 subtypes in HIV-1-seropositive samples from 333,270 residual neonatal dried blood spot samples tested for routine newborn screening tests in the UK between July 1999 and December 2002. Of the 813 antibody-positive samples shown to contain passively acquired, maternal HIV-1 for which subtyping was attempted, 333 (41%) could not be subtyped due to cross-reactivity or low values of the assay results, and 480 (59%) were classified as B (35, 7.3%) or non-B (445, 92.7%). The proportions of subtyped B samples differed significantly (P=0.004) between those from neonates whose mothers were born in the UK (21.4%) and those from neonates whose mothers were known to be born abroad (7%). Using a serological approach to establish viral serotype, we document the distribution of HIV-1 subtypes in infected pregnant women in the UK.