Aphyosemion musafirii, new species, is described from specimens collected near Ubundu (Ruiki River, Congo Basin, Tshopo Province, Democratic Republic of the Congo). Another population of A. musafirii is known from the Romée River, 50 km West of Kisangani. The Ruiki and Romée Rivers are small tributaries on the left bank of the Congo River. Aphyosemion musafirii can be distinguished from its closest relative A. castaneum by the male colour pattern. A preliminary DNA analysis demonstrates that Aphyosemion s.s. consists of two major clades. Aphyosemion musafirii is in a clade with A. castaneum, A. polli, A. lamberti, A. rectogoense, and A. congicum. The distribution of all species of Aphyosemion s.s. is discussed