In this study, three molecular marker systems including sequence related amplified polymorphism (SRAP), random amplified polymorphic DNA (RAPD), and inter-simple sequence repeats (ISSR) were screened to select polymorphisms of 24 main commercial strains of Lentinula edodes cultivated widely in China. Twenty-nine sequence characterized amplified region (SCAR) markers were developed to set up a dendrogram using UPMGA based on nucleotide sequences of some SRAP, RAPD, and ISSR polymorphic fragments. The grouping showed that the 24 strains were apparently clustered into five groups at a level of 0.68 similarity coefficient, and those that have similar breeding background clustered preferentially into the same subgroup. Results also revealed that the 24 strains had a low level of genetic diversity, and the breeding source of L. edodes should be broadened by exploiting wild types and introducing exotic strains. In addition, the tested strains of L. edodes could be clearly distinguished and identified from others by using different combinations of SCAR primers. Thus, results of this work demonstrated that SCAR was an excellent genetic marker system to characterize and investigate genetic diversity of L. edodes. Furthermore, this provided an alternative method to identify the genetic relationship of different strains of other fungi.