Peroxidase variation has been assessed in 63 populations (4322 individuals) of Acacia karroo HAYNE (Acaciae: Mimosoideae) from across its entire southern African range. The 5 bands detected in the fast zone by starch gel electrophoresis combine to form 27 different phenotypes, with between 2 and 22 phenotypes found per population. SHANNON’s measure of phenotype diversity varied from 0.15 to 4.11 (mean 2.71), whilst apportionment of the diversity showed that 81% of the diversity occurred within populations. Six coastal dune populations of A. karroo from Zululand/Mozambique clustered together and had a low phenotypic diversity. Band M is common throughout the range of the species, but it is virtually fixed to the exclusion of the other four bands in the coastal dune populations. Band K, on the other hand, is restricted to the Karoo region of the Cape Province in South Africa with lower frequencies in the adjacent highveld region of the central and western Transvaal Province to the North; it is completely absent to the east of the Drakensberg mountains and very rare in the west and north of its range. The implications of these data for seed sampling strategies in A. karroo are that although the high proportion of within-population variation makes it appropriate to sample large numbers of individuals within fewer populations to sample maximum genetic diversity, it is equally important to sample populations where bands are at high frequency because of the adaptive advantage that this may represent.