The genus Agaricus encompasses the cultivated mushroom A. bisporus, and includes both unifactorial heterothallic and homothallic species. The intractable nature of their life cycles and an absence of diagnostic morphological features, hinder analysis of breeding systems in Agaricus and some other homobasidiomycete species. We utilised RAPD markers to assess genetic variation in single spore progeny from different Agaricus species and to confirm heterokaryon formation in matings. Single spore progeny from heterothallic species should be more heterogeneous than those from homothallics. Homokaryotic progeny from the secondarily homothallic A. bisporus were less variable (12%) with fewer segregating loci than the known heterothallic species, A. bitorquis (50%) and A. nivescens (32 %). Variation in the progeny of A. campestris (36%) was similar to that for the heterothallics. Heterothallism in the field mushroom was confirmed by RAPD analysis of putative matings; the first time A. campestris heterokaryons have been constructed. No variation was observed in progeny from two collections of A. subfloccosus. Homogeneity in RAPD profiles, absence of matings and self-fertility of single spores shows that A. subfloccosus is homothallic. In progeny from A. arvensis very low variation (5%) occurred and proportions of segregating loci were around half of those for A. bisporus. However, matings were observed and RAPD characterised within the A. arvensis progeny. RAPD analysis of genetic variation within spore progeny should have general utility in the characterisation of fungal breeding systems.