Small but significant differences were found in allele frequencies among five populations (overall F ST estimate (θ)=0.004, P=0.006; overall R ST estimate (RHO)=0.019, P<0.00001) of the demersal cichlid Copadichromis sp. `virginalis kajose', collected from five locations in Lake Malawi. Pairwise F ST estimates revealed significant differences between the most southerly population (Cape Maclear), and the three most northerly populations (Mbamba Bay, Metangula and Chilola). Pairwise R ST estimates also revealed significant differences between some populations, but no geographical pattern was discernible. There was no evidence of isolation by distance using either the shortest straight-line distance between samples, or the distance around the shoreline following a 50 m depth contour. F ST, estimates were considerably lower than found in previous studies on the mbuna (rock-dwelling species), but higher than those found in a study of three pelagic cichlid species from Lake Malawi. Substructuring in C. sp. `virginalis kajose' appears to be on a similar scale to the Atlantic cod. © 2001 The Fisheries Society of the British Isles.