Prior chemical analysis of obligate thermophilic fungus Talaromyces thermophilus led to the discovery of thermolides A-F, six previously undescribed members of the lactam-bearing macrolactone class. A combination of chemical screening, genome analyses, and genetic manipulation led to the identification of the thermolide biosynthetic genes from sister thermophilic fungi T. thermophilus and Thermomyces lanuginosus and a new thermolide. The biosynthetic locus for the thermolides' mixed polyketide/amino acid structure encodes a hybrid polyketide synthase-nonribosomal peptide synthetase (PKS-NRPS). Our results reveal the first fungal hybrid iterative PKS-NRPS genes involved in the biosynthesis of bacterial-like hybrid macrolactones instead of typical fungal tetramic acids-containing metabolites. The finding provides an insight into the convergent biosynthetic end products that bridge the gap between the modular and iterative PKS-NRPS hybrids.