This study investigated the incidence and diversity of Tolypocladium within trunks of south Florida slash pines (Pinus densa). Thirty-five isolates were recovered from trunk tissue including living phloem, cambium, and sapwood. Two novel species of Tolypocladium (T. subtropicale and T. trecense) are described here based on morphological and molecular analysis of concatenated LSU, ITS, tef-1, tub, and RPB1 sequences. Our findings expand our understanding of the distribution, diversity, and ecology of this genus and confirm that it is widely spread as an endophyte across ecosystems and hosts. Strains collected in this survey will be used in future bioassays to determine their potential ecological roles as mycoparasites or entomopathogens.