In the cultivation of edible mushrooms, including Pleurotus pulmonarius (Fr.) Quel., the enormous number of spores produced by fruiting bodies can adversely affect mushroom growers' health, mushroom cultivation facilities, and the genetic diversity of natural populations. In this study, we constructed a primary genetic linkage map and identified the locus associated with the sporulation-deficient (sporeless) mutation of P. pulmonarius using 150 progeny isolates derived from a cross between sporeless and wild-type isolates. Based on the segregation of 300 AFLP markers, two mating-type factors, and the sporeless trait, a linkage map was generated consisting of 12 linkage groups. The map covered a total genetic distance of 971 cM, with an average marker interval of 5.2 cM. The gene region responsible for the sporeless mutation was located in linkage group II including 40 AFLP markers and the A mating-type factor locus. Of these markers, the nearest marker to the sporeless locus was located 1.4 cM away. Construction of this P. pulmonarius genetic linkage map and identification of markers that are closely linked to the sporeless locus will facilitate marker-assisted selective breeding of a sporeless strain with economically important traits.