We present a molecular phylogeny of African stream frogs (genus Strongylopus), based on 12S rRNA, 16S rRNA, the nuclear recombination activating gene 1 (RAG-1) and tyrosinase exon 1 (tyr). Molecular data were supported by advertisement call analysis and morphology. We recognise six valid species: Strongylopus bonaespei (Dubois, 1981) from the southern and southwestern parts of the Western Cape Province, South Africa; Strongylopus fasciatus (Smith, 1849) from eastern South Africa to Zimbabwe; Strongylopus grayii (Smith, 1849) found throughout South Africa with older records in Naukluft, in central Namibia; Strongylopus rhodesianus (Hewitt, 1933) known from the eastern highlands of Zimbabwe and western Mozambique; Strongylopus wageri (Wager, 1961) from KwaZulu-Natal Province, South Africa and Strongylopus merumontanus (Lönnberg, 1907) from eastern Zambia, Malawi, northern Mozambique and Tanzania. Strongylopus fuelleborni (Nieden, 1911), S. kitumbeine Channing & Davenport, 2002 and S. kilimanjaro Clarke & Poynton, 2005 were shown to be junior synonyms of Strongylopus merumontanus. Strongylopus springbokensis Channing, 1986 is recovered as a junior synonym of Strongylopus grayii. Divergence ages were estimated, and discussed in terms of paleoclimatic events.