The population structure of several Australian populations of the cactophilic Drosophila buzzatti was investigated, with seasonal samples from two populations analyzed for six polymorphic allozymes. Significant inbreeding throughout the year was detected in both populations, and significant but relatively slight differences in allele frequencies between collections were found in one population. Little significant 2-locus linkage disequilibrium was detected and the variances of linkage disequilibrium coefficients were generally consistent with genetic drift. Individual breeding substrates (rotting cactus cladodes) were collected and the adult flies emerging from them were scored for their allozyme genotypes. The data suggest that approximately ten individuals contribute to the progeny emerging from a rot. The influence of the sizes and distributions of breeding substrates on the maintenance of genetic variation is discussed.