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The taxonomy and phylogeny of the genus <i>Tolypocladium</i> are herein revised based on the most comprehensive dataset to date. Two species-level phylogenies of <i>Tolypocladium</i> were constructed: a single-gene phylogeny (ITS) of 35 accepted species and a multigene phylogeny (nr<i>SSU</i>, nr<i>LSU</i>, <i>tef</i>-<i>1α</i>, <i>rpb1,</i> and <i>rpb2</i>) of 27 accepted species. Three new species, <i>Tolypocladium pseudoalbum</i> sp. nov., <i>Tolypocladium subparadoxum</i> sp. nov., and <i>Tolypocladium yunnanense</i> sp. nov., are described in the present study. The genetic divergences of four markers (ITS, <i>tef</i>-<i>1α</i>, <i>rpb1</i> and <i>rpb2</i>) among <i>Tolypocladium</i> species are also reported. The results indicated that species of <i>Tolypocladium</i> were best delimited by <i>rpb1</i> sequence data, followed by the sequence data for the <i>rpb2</i>, <i>tef</i>-<i>1α</i>, and ITS provided regions. Finally, a key to the 48 accepted species of <i>Tolypocladium</i> worldwide is provided.