China is home to rich wild and cultivated strains of Lentinus edodes, an important edible and medicinal mushroom. Artificial selection of L. edodes has a long history, and the widely cultivated strains belong to populations different from those of most wild strains. Internal transcribed spacer (ITS) regions have been used as good markers to identify L. edodes populations. But because ITS regions exhibit incomplete concerted evolution, the use of an ITS to identify L. edodes populations has been questioned. The objective of this study was to determine whether the ITS region is suitable for identifying L. edodes populations and which populations the widely cultivated strains and the most wild strains belong to by investigating intraindividual and differential ITS polymorphisms between 44 cultivars and 44 wild strains of L. edodes in China. Intraindividual ITS polymorphism is common in L. edodes strains, and most strains possessed 2 different ITS sequences, which came from their heterokaryons. The genetic polymorphisms of ITS1, 5.8S, and ITS2 in L. edodes strains are distinct. All strains were divided into one 5.8S type (5.8S-A), 2 ITS1 types (ITS1-A and ITS1-B), and 2 ITS2 types (ITS2-A and ITS2-B), which were subdivided into 2 branches (ITS2-A1 and ITS2-A2; ITS2-B1 and ITS2-B2). ITS1/5.8S/ITS2 could be used as a good marker in preliminary classification of L. edodes strains in China. It not only exhibited classified information of ITS1, 5.8S, and ITS2 for each strain at the same time, it also indicated whether the strain was heterozygous. The 44 cultivated strains were mainly the A/A/A1 type, and the 44 wild strains were mainly the A/A/A2 and other mixed types.