To characterize the differences between kDNA minicircles of drug-resistant Leishmania mexicana amazonensis variants that show nuclear DNA amplification and minicircles of variants without nuclear DNA amplification, we sequenced minicircles from repeatedly cloned parasites. The dominant minicircles from arsenite- and tunicamycin-resistant parasites with DNA amplification were found to preexist as minor conserved divergent classes in parental wild-type cells. These classes shared very limited similarity with the predominant wild-type minicircle sequences or sequences from drug resistant parasites without amplification. These minor classes were preferentially selected to replicate in variants with DNA amplification and subsequently became the dominant sequences in these variants. Kinetic studies of the correlation between amplification and deamplification of the nuclear DNA and the switch in kDNA minicircle dominance indicated that factor(s) other than the amplified chromosomal DNA itself caused the minicircles to switch. Treating the kDNA networks isolated from cells at the switch transition period with single cutter endonucleases specific for either wild-type or variant-specific minicircles resulted in structural modifications consistent with both minicircle sequence classes being present simultaneously in the same network. This establishes the 'trans' nature of the switch.