The secondarily homothallic life cycle of white button mushroom which results in scarcity of uninucleate basidiospores (homokaryons) in its progeny, is the most impediment for genetic improvement of its strains. With respect to uncertainty of fruiting test and nonexistence of specified morphological criterion, in two recent decades scientists have used molecular markers as a fast and certain criterion for screening of homokaron isolates. In this study, RAPD markers have been used for identification of homokaryon isolates. Results showed that RAPD markers could discriminate homokaryons and heterokaryons based on number of bands. The number of band in homokaryons significantly reduced compared to those of heterokaryons. Results also showed that cluster analysis based on average of band number could separate homokaryon and heterokaryon isolates in two distinct groups.