Tolypocladium guangdongenseis a high-value edible fungus with various medicinal and food safety properties. However, its evolutionary and genetic information is still limited. Mitochondrial genomes are potential models for molecular evolution and phylogenetic studies. In this study, we sequenced the complete mitogenome ofT. guangdongense, demonstrating circular sequence of 46,102 bp, containing 14 standard protein-coding genes (PCGs), 2 ribosomal RNA subunit genes, and 28 tRNA genes. Phylogenetic analysis based on mitochondrial genes indicated thatT. guangdongensewas clustered into theTolypocladiumgenus with high support value, based on the core PCG dataset. In addition,rps3is also a suitable marker in the phylogenetic analysis inHypocreales. Gene rearrangement analysis indicated that the gene order of PCGs was highly consistent inHypocreales, and tRNA rearrangement events occurred in most species ofHypocreales; however, the rearrangement rates were not taxonomically correlated. Divergence time estimation based on the old fossil record and previous reports revealed thatT. guangdongenseoriginated approximately in the middle Cenozoic (42 Mya, 95% highest posterior density interval: 43-116) with theTolypocladiumgenus differentiation. Our results provided more mitogenomic information ofT. guangdongenseand shed new insights into evolution of theTolypocladiumgenus.