The relationships between relative mRNA expression levels of the large ribonuclease reductase subunit (RNR-LC) and small subunit (RNR-M2) genes and cordycepin content in different Cordyceps militaris strains were determined and analyzed to investigate the transcriptional regulation of the cordycepin synthesis pathway by RNR. Cordycepin content in various C. militaris strains was measured, and quantitative analysis of mRNA expressed by the RNR-LC and RNR-M2 genes was performed by quantitative fluorescent PCR with the use of two internal control genes for experimental data correction. Wild C. militaris, which had the lowest cordycepin content, was used as control. Results indicated that mRNA expression levels of the RNR-LC and RNR-M2 genes in the rice-cultivated C. militaris group were 1.28 and 1.47 times that of the control group, respectively. The corresponding mRNA expression levels of the silkworm chrysalis-cultivated C. militaris group were 2.35 and 3.84 times that of the control group, respectively. Compared with the control group, the mRNA expression level of RNR-M2 generally increased with an increase in cordycepin content. There were significant differences in the relative expression levels of both genes among different C. militaris strains (p<0.05). Obvious dominant expression of the RNR-M2 gene was observed in strains with high cordycepin content, while the expression level of the RNR-LC gene did not change significantly with cordycepin content. Therefore, a synergistic regulatory role does not exist between the two genes. It can be deduced that the RNR-M2 gene plays a key role in the positive regulation of the cordycepin synthesis pathway.