In this study, we have investigated the diversity of the current HIV-1 strains circulating in Addis Ababa, Ethiopia; in addition, we have evaluated the applicability of peptide enzyme-linked immunosorbent assay (ELISA) and heteroduplex mobility assay (HMA) for HIV-1 subtyping. Previous studies have indicated that HIV-1 subtype C is the major subtype present in HIV-positive samples collected from various risk groups between 1988 and 1995 in Addis Ababa. To assess the possible influx of new HIV-1 subtypes, 150 commercial sex workers (CSW) reporting in 1997 to two Health Centers in Addis Ababa were enrolled in an unlinked anonymous cross-sectional study. Subtyping was performed according to the World Health Organization algorithm of peptide ELISA, followed by HMA and DNA sequencing. As a result, the HIV-1 prevalence among these CSWs was found to be 45% (67 of 150). Of the 67 samples, 66 contained HIV-1 of subtype C and only one was of subtype D. This confirms the persistent overall presence of HIV-1 subtype C in Addis Ababa and a low influx of other subtypes into this location.