Abstract Pheromone and pheromone receptor genes in B mating type locus constitute B mating type of fungi belonging to Basidiomycota. In Lentinula edodes, multiple B mating types have been suggested based on genome sequence analysis and mating assay. Here we report finding of new alleles of pheromones (phbs) and pheromone receptors (rcbs) to constitute five alleles of rcb1 (rcb1-1∼rcb1-5) with nine associated phbs in Bα sublocus and three alleles of rcb2 (rcb2-1∼rcb2-3) with five associated phbs in Bβ sublocus. Each rcb was primarily associated with two phbs. Each phb-rcb pair was recurrently discovered as a distinct unit in various monokarytotic and dikaryotic strains, regardless of subloci. This allowed us to propose 15 B mating types through combinations of five Bα and three Bβ subloci. PCR analyses with primer sets probing alleles of rcb1 and rcb2 demonstrated that all monokaryotic strains contained one of the five rcb1s and one of the three rcb2s representing Bα and Bβ subloci, respectively, thereby enabling designation of their B mating types via combinations of Bα and Bβ. PCR analyses also revealed the presence of one or two alleles of rcb1 and rcb2 representing B mating type of each nuclei in dikaryotic cytoplasm. Further investigation of 111 dikaryotic strains, including 83 cultivated and 28 wild strains collected from East Asia, revealed that B8 and B11 mating types constructed by rcb1-3 + rcb2-2 and rcb1-4 + rcb2-2, respectively, were more prevalent in cultivated strains. In B mating type pairs representing each nucleus in dikaryons, B2B12, B4B8, and B8B11 were frequently occurring pairs in cultivated strains while none of them was found in wild strains. Such prevalence indicates that certain nuclei have been preferentially selected in the generation of cultivated strains. Our findings shed new light on the construction of B mating type in L. edodes and provide practical tools in the breeding of new cultivars.