[Objective] To analyze the molecular evolution of three protein-coding genes (csp1, MAT1-1-1 and MAT1-2-1) among various Ophiocordyceps sinensis isolates. [Methods] We amplified partial csp1 fragment and complete MAT1-1-1 and MAT1-2-1 gene sequences from 125 broadly sampled O. sinensis isolates, and compared the topological difference of phylogenetic trees constructed by exons and introns, and of those constructed by MAT1-1-1 and MAT1-2-1. We also measured selection pressures and detected evidence of recombination for the three genes. [Results] The length of exons of the three protein-coding genes was conserved among different O. sinensis isolates with 4.5%?5.7% of variable sites. The length of introns was either identical or different among different isolates with more than 1.8%?22% of variable sites. For the two mating-type genes, MAT1-1-1 was less variable than MAT1-2-1. Phylogenetic topology differences were significant between exons and introns, and between MAT1-1-1 and MAT1-2-1 exon sequences. Each of the three genes was undergoing purified selection. Recombination events were detected between different DNA sites within each gene, but not obvious between different gene fragments. [Conclusion] Because different gene fragments and different regions of a gene in O. sinensis might be evolutionarily different, we should use multiple gene fragments in future evolution-related studies in O. sinensis.