Ophiocordyceps sinensis (OS) has a variety of pharmacological effects and has been widely used as tonic food and medicine for hundreds of years. Among them, the OS of Yushu in Qinghai and Naqu in Tibet are considered to be of high quality. However, a lack of evidence on the difference in metabolites between OS from two typical growing regions. We compared the metabolomes of OS from Qinghai and Tibet through a comparative metabolomics approach based on ultra performance liquid chromatography coupled with triple quadrupole linear ion-trap tandem mass spectrometry. A total of 646 metabolites were identified from the OS of two typical growing regions, and there were no differences in the types of metabolites of OS in different regions, but in the differences in contents. Among the 85 metabolites screened with significant differences, cyclic peptides had a high content in Tibet group, while nucleotides, organic acids and their derivatives and lipids had a high content in Qinghai group. Metabolic pathways analysis of differential metabolites revealed that “vancomycin resistance”, “tryptophan metabolism”, “serotonergic synapse”, “phenylalanine metabolism”, “neomycin, kanamycin and gentamicin biosynthesis”, and “insulin resistance” were the main enrichment pathways. These findings provide an insight into the differential metabolic profiling and metabolic pathways of OS from two typical growing regions.