Background: Pleurotus tuoliensis (Bailinggu) is a commercially cultivated mushroom species with an increasing popularity in China and other Asian countries. Commercial profits are now low, mainly due to a low yield, long cultivation period and sensitivity to diseases. Breeding efforts are thus required to improve agronomical important traits. Developing saturated genetic linkage and physical maps is a start for applying genetic and molecular approaches to accelerate the precise breeding programs. Results: Here we present a genetic linkage map for P. tuoliensis constructed by using 115 haploid monokaryons derived from a hybrid strain H6. One thousand one hundred and eighty-two SNP markers developed by 2b-RAD (type IIB restriction-site associated DNA) approach were mapped to 12 linkage groups. The map covers 1073 cM with an average marker spacing of 1.0 cM. The genome of P. tuoliensis was de novo sequenced as 40.8 Mb and consisted of 500 scaffolds (>500 bp), which showed a high level of colinearity to the genome of P. eryngii var. eryngii. A total of 97.4% SNP markers (1151) were physically localized on 78 scaffolds, and the physical length of these anchored scaffolds were 33.9 Mb representing 83.1% of the whole genome. Mating type loci A and B were mapped on separate linkage groups and identified physically on the assembled genomes. Five putative pheromone receptors and two putative pheromone precursors were identified for the mating type B locus. Conclusions: This study reported a first genetic linkage map integrated with physical mapping of the de novo sequenced genome and the mating type loci of an important cultivated mushroom in China, P. tuoliensis. The de novo sequenced and annotated genome, assembled using a 2b-RAD generated linkage map, provides a basis for marker-assisted breeding of this economic important mushroom species.