Aspergillus fumigatusis one of the most common pathogenic fungi, which results in high morbidity and mortality in immunocompromised patients. Amphotericin B (AMB) is used as the core drug for the treatment of triazole-resistantA. fumigatus. Following the usage of amphotericin B drugs, the number of amphotericin B-resistantA. fumigatusisolates showed an increasing trend over the years, but the mechanism and mutations associated with amphotericin B sensitivity are not fully understood. In this study, we performed ak-mer-based genome-wide association study (GWAS) in 98A. fumigatusisolates from public databases. Associations identified withk-mers not only recapitulate those with SNPs but also discover new associations with insertion/deletion (indel). Compared to SNP sites, the indel showed a stronger association with amphotericin B resistance, and a significant correlated indel is present in the exon region ofAFUA_7G05160, encoding a fumarylacetoacetate hydrolase (FAH) family protein. Enrichment analysis revealed sphingolipid synthesis and transmembrane transport may be related to the resistance ofA. fumigatusto amphotericin B. The expansion of variant types detected by thek-mer method increases opportunities to identify and exploit complex genetic variants that drive amphotericin B resistance, and these candidate variants help accelerate the selection of prospective gene markers for amphotericin B resistance screening inA. fumigatus.