The HIV-1 epidemic in Tanzania is characterized by the circulation of heterogeneous virus subtypes. A retrospective study was conducted to determine the changing pattern of circulating HIV-1 subtypes in northern Tanzania. A peptide-binding enzyme immunoassay (PEIA) was employed to analyse 305 HIV-1 positive serum and plasma samples collected between 1985 and 2005. Samples were serotyped using synthetic peptides representing HIV-1 genotypes A-E derived from consensus gp120 V3 sequences. Plasma samples collected in 2005 were V3 genotyped for comparison with PEIA results. In 1985, serotypes A and D were co-circulating while serotype C was first detected in 1990. In 2001 and 2005, serotype C was the most prevalent, serotype A was stable, and serotype D was declining. PEIA is relatively rapid and simple to perform compared to molecular approaches, and is a valuable epidemiological tool in regions with limited resources. HIV-1 classification into serotypes based on antigenic V3 diversity may be a useful screening tool for the identification of HIV-1 variants with regard to diagnosis, treatment, disease progression and candidate vaccine development.