A crown and root rot complex was detected in the alfalfa (Medicago sativa 'Longdong') fields of Huanxian County. The symptoms of the diseased plants were characterized, and 11 fungal species were obtained from the roots. These fungi included isolates that resembled the genus Microdochium. An isolate of this type, designated MP313, was proven to infect alfalfa, fulfilling Koch's postulates. Isolate MP313 was examined by microscopy and the morphological characteristics indicated that it was similar to members of the genus Microdochium. Sequence analyses of the 28S large subunit as well as the internal transcribed spacer (ITS) region of the ribosomal DNA (rDNA) of MP313 revealed 98 to 100% similarity to the corresponding regions of M. tabacinum. A polymerase chain reaction assay based on the ITS region of the rDNA was developed to amplify a 304-bp fragment from DNA concentrations as low as 20 fg/μl, which was sensitive enough to detect isolate MP313 in diseased root samples. Taken together, these results confirmed that M. tabacinum was one of a complex of fungi associated with crown and root rot in the alfalfa samples collected in Gansu Province. This is the first report of M. tabacinum being a pathogen of alfalfa in China.