There is close relationship between the mating type gene of Ophiocordyceps sinensis and the artificial introduction of fruiting body. Many scholars focus on the mating type of O.sinensis, and MAT1-2-1 sequences of 40 isolates of O.sinensis by different methods were amplified with the primers MAT1-2-1 and evolutionary phylogenetic analyses were performed with sequences of the MAT1-2-1 gene, of which 7 sequences of MAT1-2-1 came from this experiment randomly and 4 sequences of MAT1-2-1 cited from the GenBank data. The result showed that 7 isolates collected from Qinghai, together with the isolates from Qinghai in GenBank make up a large group with the 96% bootstrap value and the isolates from the other area clustered into the other part. Meanwhile, all of single ascospore isolates of O.sinensis contain the MAT1-2-1 gene and the ratio of MAT1-1: MAT1-2 has not been found by 4:4. So O. sinensis might not be heterothallism, to a large extent, it is homothallic.