Long-eared bats of the genus Plecotus are widespread over most of temperate Eurasia, marginally reaching the African continent and Macaronesia. Previously, all African populations were assigned to one species, P. auritus, and later to P. austriacus. We analysed museum specimens of African long-eared bat populations using both morphologic and genetic techniques. Based on morphological evidence we recognise four well-defined allopatric populations in northern Africa. They differ in fur coloration, skull morphology and bacular traits. The molecular data support a division of the African populations into at least three well-separated evolutionary lineages. With a combination of these data we define three species of Plecotus occurring in Africa (incl. the Canary Islands) and describe a new subspecies. Small, very pale greyish-brown Egyptian long-eared bats (P. christii Gray, 1838) inhabit desert and semi-deserts habitats of eastern Sahara (Libyan Desert, Nile Valley of Egypt and northern Sudan). Smaller to medium-sized, dark brown Ethiopian long-eared bats (P. balensis Kruskop et Lavrenchenko, 2000) inhabit the Ethiopian Highlands above 2000 metres a. s. l. This form represents the only Afro-tropical species of Plecotus. Large, dark greyish Canarian long-eared bats (P. teneriffae teneriffae Barret-Hamilton, 1907) occur on the three western islands of the Canarian Archipelago. A medium-sized greyish-brown Gaisler’s long-eared bat, P. teneriffae gaisleri subsp. n., is described from the Mediterranean region of Cyrenaica, north-eastern Libya. Due to the lack of substantial morphological differences we preliminarily consider the Maghrebian population of long-eared bats to be consubspecific with P. teneriffae gaisleri subsp. n. The systematic position of the population of Cape Verde Islands remains uncertain.