Agrocybe salicacola is a delicious and cultivable mushroom. It is important to understand this species' inherent characteristics, especially to elucidate the constitution and segregation of mating genes. In this study, two compatible B mating loci in strain YAASM0711 of A. salicacola were cloned from the monokaryons, and sequence and phylogeny analyses showed two conserved genes encoding pheromone receptors maybe lost mating activity, which determined by comparing with those of other mushrooms. In the conserved regions of mating loci, partial insertion/deletion fragments made non-coding regions posses polymorphisms, and monokaryotic strains of different mating types were distinguished from each other according to the amplification profile of variable regions, which suggested mating loci were integrally assigned to offspring strains during mitosis in A. salicacola. As our known, it is the first to develop molecular markers for B mating-type identification using variable non-coding fragments of mating loci in basidiomycetes.