The A421 ABCG2 genotype is a frequent polymorphism encoding the K141 transporter, which is associated with a significant decrease in transporter expression and function when compared to the wild type (wt) C421 allele encoding the Q141 ABCG2. Here we show that during the acquisition of resistance to the novel triazoloacridone antitumor agent C-1305 in lung cancer cells harboring a heterozygous C421A genotype, a marked C421 allele-specific ABCG2 gene amplification occurred. This monoallelic C421 ABCG2 gene amplification brought about the overexpression of both C421 ABCG2 mRNA and the transporter at the plasma membrane. This resulted in the lack of cellular drug accumulation due to increased efflux of both C1305 and C-1311, a fluorescent imidazoacridone homologue of C-1305, as well as marked resistance to these antitumor agents and to established ABCG2 substrates including mitoxantrone and SN-38. Consistently, the accumulation and sensitivity to these drugs were restored upon incubation with the potent and specific ABCG2 transport inhibitors Ko143 and fumitremorgin C. Moreover, upon transfection into HEK293 cells, the wt Q141 ABCG2 allele displayed a significantly decreased accumulation of C-1311 and increased resistance to C-1305, C-1311 and mitoxantrone, when compared to the K141 ABCG2 transfectant. Hence, the current study provides the first evidence that during the exposure to anticancer drugs, an allele-specific Q141 ABCG2 gene amplification occurs that confers a drug resistance advantage when compared to the K141 ABCG2. These findings have important implications for the selection and expansion of malignant anticancer drug resistant clones during chemotherapy with ABCG2 drugs.