The diversity of Agrocybe aegerita multispecies complex in Yunnan Province, southwest China was investigated. In total, 23 strains collected from ten districts were classified into three groups through mitochondrial small-subunit (mtSSU) rDNA sequence analysis. Phylogeny of internal transcribed spacer (ITS) combined with mating tests demonstrated that the tested strains included at least two species. Agrocybe salicacola, which is difficult to distinguish from other species in the A. aegerita complex, was first systematically analyzed and identified with molecular characterization. YAASM0594 and YAASM0625 share sequences with YAASM1024, and XW01 in group B, defined according to the mtSSU rDNA sequences, was related more closely to group C in the ITS phylogenetic tree, indicating that group B and CS45 are closely related, with both groups possibly even being the same species. We conclude that a high diversity occurs in Agrocybe aegerita complex than reported previously, and that more novel species would possibly be discovered through molecular phylogeny and mating tests.