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Interspecies hybridization is considered as the most important tools for improving genetic characteristics of <em>Agaricus bisporus</em>, which require having compatible homokaryons to cross. One challenge is low percentage of homokaryons among the basidiospores because of the secondary homothalism of this mushroom. Therefore, selection and confirmation of homokaryons among single spore isolates has always been involved with laborious efforts and researchers struggle with it. In this study, we attempted to develop a high-throughput method with high accuracy for screening and confirmation of homokaryons. We used 10 SSR markers -that represented nine linkage map groups of <em>Agaricus bisporus</em>-. Of 71 isolates, several isolates showed two bands as the same as those that heterokaryotic parents did, while some of them showed only one band. We were able to divide the isolates into two main groups: homoallelic and heteroallelic. The homoallelic group included 23 isolates that were monoallelic in the whole sites; which were thus confirmed as homokaryons. Further, the findings of the molecular markers were compared to the observations of a fruiting test. The results revealed that isolates that were confirmed by SSR to be heterokaryotic did not produce fruit bodies in the fruiting test. These findings obviously suggested that fruiting tests are less reliable than SSR markers. Genetic variation among 23 putative homokaryons was also calculated with the NTSYSpc software. The genetic similarity was variable between 0.3-1 among the isolates. The isolates were divided into two main groups by dendrogram. The results showed that these 10 SSR markers are able to detect homokaryons with a probability rate over 99 percent.