Polyoxins are fungicides that inhibit the synthesis of a cell wall polysaccharide called chitin. Strains resistant to polyoxins have emerged in several fungi. However, the mechanisms responsible for polyoxin resistance are not well understood. In this study, we used chemical mutagenesis and a genetic screening approach in Bipolaris maydis to elucidate said mechanisms. Two polyoxin-resistant strains were obtained (PR2027 and PR2029). Through crossing experiments, it was concluded that polyoxin resistance in these strains was caused by an alteration at a single locus. Whole genome comparison and linkage analysis were used to determine the causative mutations: the candidate causative mutation for PR2027 was in the gene coding for the translation initiation factor 3, subunit h (eIF-3h); the candidate causative mutation for PR2029 was in the gene coding for the RNA polymerase III, second largest subunit. To confirm whether the identified mutation was responsible for phenotypic alteration, the mutant eIF-3h-gene was ectopically inserted to the flanking region of HIS3 in the wild-type strain. The resulting strain (eIF-3hWM) did not show polyoxin resistance. However, after disrupting the wild-type allele of eIF-3h in the original locus of eIF-3hWM, the colony became resistant to polyoxins. Furthermore, ectopic introduction of the wild-type eIF-3h gene into the flanking region of HIS3 in PR2027 altered its phenotype from resistant to susceptible. These results suggest that the mutation in the gene encoding eIF-3h is responsible for polyoxin resistance in PR2027. Confirmation of the proposed causative gene in PR2029 is still ongoing.