Abstract: ITS sequences are commonly used molecular markers and are widely used in fungal phylogeny. However, there is a lack of comprehensive analysis on groups of Cyclocybe aegerita. The findings indicated that Intra-strain ITS heterogeneity with positional double peaks was identified in 8 strains, and each of them contained two types of ITS sequences based on the sequence analysis of monokaryons, suggesting these heterozygotes probably produced by the cross of monokaryons from different groups. Cloning of ITS products from heterozygotes produced some chimeric sequences, and the template switching occurs in ITS1 and ITS2 regions. All strains in phylogenetic tree constructed by mtSSU and ITS sequences, but the topology structure of them was not accordant. Additionally, heterogous ITS derived from the same strain showed obvious divergence even falling in different groups. Our study showed the diversity and complexity of the C. aegerita strains, and it suggests the direct sequencing of ITS products from heterozygotes probably produce wrong result on the strain identification and phylogenetic analysis of the C. aegerita species complex.