Various gene alterations related to acute leukemia are reportedly involved in drug resistance. We investigated Idarubicin (IDR) resistance by using exome nuclear DNA analyses of the human acute leukemia cell line MOLT-3 and the derived IDR-resistant cell line MOLT-3/IDR. We detected mutations in MOLT-3/IDR and MOLT-3 by using both GATK and SnpEff. We found 8,839 genes with specific mutations in MOLT-3/IDR and 1,162 genes with accompanying amino acid mutations. The 1,162 genes were identified by exome analysis of polymerase-related genes using the KEGG, and among these, we identified genes with amino acid changes. In resistant strains, LIG and helicase plurality genes showed amino acid-related changes. An amino acid mutation was also confirmed in polymerase-associated genes. GO enrichment testing was performed and lipid-related genes were selected from the results. Moreover, FACS was used to determine whether IDR permeability was significantly different in MOLT-3/IDR and MOLT-3. The results showed that an IDR concentration of 0.5 μg resulted in slow permeability in the case of MOLT-3/IDR. This slow IDR permeability might be because of the effects of the amino acid changes found in polymerase- and lipid-associated genes.