The opportunistic mold Fusarium solani is intrinsically resistant to cell wall synthesis-inhibiting echinocandins (ECs), including caspofungin and micafungin. Mutations that confer acquired EC resistance in Saccharomyces cerevisiae and other normally susceptible yeast species have been mapped to the Fks1 gene; among these is the mutation of residue 639 from Phe to Tyr (F639Y) within a region designated hot spot 1. Fks1 sequence analysis identified the equivalent of Y639 in F. solani as well as in Scedosporium prolificans, another intrinsically EC-resistant mold. To test its role in intrinsic EC resistance, we constructed Fks1 hybrids in S. cerevisiae that incorporate F. solani hot spot 1 and flanking residues. Hybrid construction was accomplished by a PCR-based method that was validated by studies with Fks1 sequences from EC-susceptible Aspergillus fumigatus and paired EC-susceptible and -resistant Candida glabrata isolates. In support of our hypothesis, hybrid Fks1 incorporating F. solani hot spot 1 conferred significantly reduced EC susceptibility, 4- to 8-fold less than that of wild-type S. cerevisiae and 8- to 32-fold less than that of the same hybrid with an F639 mutation. We propose that Fks1 sequences represent determinants of intrinsic EC resistance in Fusarium and Scedosporium species and, potentially, other fungi.