Genetic variation within and between eighteen isolates of Alternaria brassicicola, five isolates of A. alternata, and a single isolate of Rhynchosporium secalis was compared using Amplified Fragment Length Polymorphisms (AFLPs). The AFLPs consistently distinguished between the three species. AFLPs thus provide a reliable tool for identifying A. brassicicola. The analysis of eighteen isolates of A. brassicicola from different sites also indicated at least moderate levels of genetic diversity within A. brassicicola along the New South Wales coast. Of 43–66 markers scored per primer combination, 16·7–27·9% were polymorphic. On the basis of an unweighted paired group method of arithmetic averages analysis using data from four primer combinations, most isolates were identified as separate genotypes. However, multiple isolates from particular locations tended to cluster together, implying the potential for population structure, and there was a significant positive correlation between Nei's measure of genetic distance and physical distance (r = 0·5486, P<0·001). Despite the absence of an identified sexual stage, A. brassicicola would appear to have a means for generating and maintaining significant variation.