The anthracenedione, mitoxantrone, frequently selects for a unique drug resistance phenotype that is not mediated by either MDR 1, MRP, or altered DNA topoisomerase II. In this study, we demonstrate that mitoxantrone resistance is likely to be multifactorial with at least one resistance mechanism being the result of a dominant genetic event. This finding was demonstrated by conducting chromosome transfer experiments from human breast cancer cell lines that were either sensitive (MCF7/S) or resistant to mitoxantrone (MCF7/Mitox). Chromosomes transferred from MCF7/Mitox cells into CHO-K1 cells resulted in the isolation of multiple clones resistant to mitoxantrone. In contrast, chromosomes transferred from the drug sensitive MCF7/S, parent cell line did not confer drug resistance in the rodent CHO-K1 recipient cell line. Both Alu-PCR analysis and Southern blot analysis demonstrated human DNA in the CHO-K1 cells receiving chromosomes from the MCF7/Mitox cells. Unlike the MCF7/Mitox cell line, the drug resistant, CHO-K1 chromosome transferrant clones did not have a decrease in total drug accumulation. We conclude that chromosome transfer from the MCF7/Mitox cell line into CHO-K1 cells, confers a non-transport mediated mechanism of drug resistance that is a dominant genetic event. These studies provide evidence of the genetic multifactorial nature of multidrug resistance in cells selected with mitoxantrone in-vitro.