Agaricus subrufescens is a cultivated edible and medicinal mushroom. Its known geographical distribution encompasses the Americas, Europe, Oceania, and Asia. The objective of this study was to assess mating compatibility and interfertility of strains originating from Brazil, France, and Thailand. Progeny of each strain were analyed with codominant molecular markers. Multilocus genotype tests revealed that the three strains were amphithallic with percentages of heterokaryotic single spore progenies of 75 % for the Thai strain and around 40 % for the Brazilian and French strains. In mating tests A. subrufescens had a multiallelic unifactorial system of sexual incompatibility. The three parent strains were interfertile based on experimental pairings of single-spore isolates, the recovery of hybrid heterokaryons from compatible matings, and the ability of hybrids to produce mushrooms and fertile spores. This biological approach supports the inclusion of the European strains within the species and the extension of the geographical distribution range to Asia. Our data should help to develop breeding strategies and to better manage and exploit the diversity existing in A. subrufescens.