Lentinula edodes is an important cultivated mushroom in China. The development of Lentinula edodes production promotes more studies on it. In our previous work, degenerate PCR and chromosome walking technologies were used to obtain one pheromone receptor gene and one pheromone precursor gene from Lentinula edodes. In this study, four pairs of specific primers were designed according to the whole genome sequencing of the protoplast monokaryon of Lentinula edodes strain 135, to amplify STE3-like pheromone receptor gene and its flanking conserved genes in the protoplast monokaryon strain SUP2 derived from Lentinula edodes strain Suxiang and 33655bp DNA sequence was obtained. By BlastX search, seven putative genes were identified, and three of them are pheromone receptor encoded genes. Furthermore, near to two pheromone receptor genes, four genes encoding proteins with conserved motifs of pheromone precursors were found. This study firstly reveals the molecular organization of the B mating type locus of Lentinula edodes.