The problems in precise discrimination of monokaryotic isolates of edible fungi hindered the breeders to enhance the improvement cycle of mushroom strains in an effective manner. In current study, 5′ end of intergenic spacer 2 (IGS2) region was amplified and used to assist categorizing the selected monokaryotic isolates of Pleurotus pulmonarius into two distinct groups (type A and B). Moreover, the dikaryotic isolates were able to produce double-banded PCR product and the monokaryons create only one unique amplicon. Candidate region approach was used in this study and enabled pedigree history to be recorded, and thus breeders are able to make informative decisions. Hence, this approach promotes time saving and breeding space to be allocated for strain improvement programmes. Meanwhile, this highly variable DNA marker can be used for the confirmation of successfully crossed strains. Eventually, this methodology can assist towards detecting efficient mating partners of P. pulmonarius in any breeding programmes including selective breeding, production of high yield hybrids and studies on structure mating types.