Abstract: Venturia inaequalis, the causal agent of apple scab, is one of the most important diseases of apple (Malus domestica), in terms of economic crop losses, world-wide and in South Africa. South Africa has four principal climatically different apple growing regions. Apple scab isolates were characterised molecularly from four populations from the different regions to determine if there are any differences between these populations. In the 2012/2013 season, single spored isolates were genotyped with six known microsatellite markers from V. inaequalis,EmVi10, Vitc2/D, Vitg9/129, Vitg11/70, 1tc1g and 1tc1a. Species identity was confirmed by using ITS and ABC2 sequencing. Results from the genotyping with the microsatellite markers indicate that the South African V. inaequalis populations are highly variable and sexually outcrossing (Vo/Ve = 1.18; P = 0.55). Overall, SSR markers used in this study were highly variable (H = 0.62). In addition, each region has ‘private’ alleles indicating moderate differences between the populations (Fst = 0.15; P = 0.001). Little population differentiation was found between the Ceres and the two Langkloof populations although these populations are more than 500 km apart. There were moderate differences between the other populations. This may have implications on the apple scab control and indicates that different management practices may be needed to control the disease in these different areas. The disease might also have adapted to the different climatic conditions in these areas. A high number of synonymous changes was found in the ABC2 gene nucleotide binding site 2 (NB2) region (TD = 0.034; P > 0.1) and six ABC2 haplotypes and five ITS haplotypes were identified. The ABC2 NB2 region might be under balancing selection, implying possible virulence differences in the pathogen.