In this study, SCAR marker that differentiates Pleurotus eryngii strains with higher β-glucan from control strain was developed. Genomic DNAs of 9 control strains of Pleurotus eryngii and 9 Pleurotus eryngii strains with higher β-glucan were analyzed by bulked segregant analysis (BSA) using randomly amplified polymorphic DNA (RAPD). One-hundred twenty RAPD primers were screened on bulked DNA samples and a unique DNA fragment with the size of 91 bp was yielded by OP-R03 primer from the Pleurotus eryngii strains with higher β-glucan. A sequence characterized amplified region (SCAR) marker, designated as OP-R03-1-F and OP-R03-1-R, was designed on the basis of the determined sequence. The PCR analysis with the OP-R03-1 primer showed that this SCAR marker can clearly distinguish the Pleurotus eryngii strains with higher β-glucan from the control strains.