Pleurotus eryngii, an edible white-rot fungus, is widespread in Eurasia and northern Africa. It has become a major cultivated mushroom in Asia, with a current global production rate of approximately 3 × 10 5 metrictons/yr. To improve the quality or productivity through breeding, a genetic linkage map is an important component. In this study, genetic linkage map of the P. eryngii was constructed using 98 monokaryotic progeny derived from dikaryon of parental KNR2312 strain derived from haploid meiotic spores. The whole genome sequence of P5 monokaryon from P. eryngii KNR2312 strain by Next Generation Sequencing (NGS) strategy was used to design the SSR markers. 484 primers pairs were identified by SSR Locator I and tested polymorphism via PCR. A total of 241 loci were mapped using Joinmap 4.0, comprising 222 SSR markers, 2 mating type factors, and the 13 INDEL markers. The map consisted of 14 linkage groups spanning 1003 cM at an average marker interval of 4.2 cM. The mating loci, A and B were mapped on linkage groups 4 and 11, respectively. The established linkage map and the genetic information based on NGS could be used for QTL mapping of agronomic traits, marker-assisted breeding that may ultimately lead to outstanding phenotypic characteristics. [Supported by a grant from the IPET (213003-04-3-SBY20), MIFAFF, Republic of Korea.]