The release and spread of a large number of basidiospores from the developing fruiting body cause serious problems in the cultivation of edible mushrooms, including Pleurotus pulmonarius (Fr.) Quel. The P. pulmonarius sporeless mutant, TMIC-30058, has a high potential for breeding sporeless cultivars to reduce these adverse effects. Our previous study (Okuda et al. in Breeding Science 59:315–319, 2009) showed two sequence-tagged site (STS) markers for the sporeless trait from this mutant. Here, using these STS markers we present rapid detection of sporeless trait from P. pulmonarius culture extracts by real-time PCR. This method enables us to cut down time and labor for screening of the sporeless trait, suggesting its availability for efficient breeding of sporeless cultivars.