The molecular mechanisms by which the malarial parasite has managed to develop resistance to many antimalarial drugs remain to be completely elucidated. Mutations in the pfmdr1 gene of Plasmodium falciparum, as well as an increase in pfmdr1 copy number, have been associated with resistance to the quinoline-containing antimalarial drugs. We investigated the mechanisms of drug resistance in Plasmodium using a collection of P. yoelii lines with different drug resistance profiles. The mdr1 gene of P. yoelii (pymdr1) was identified and characterized. A 2- to 3-fold increase in the pymdr1 gene copy number was observed in the P. yoelii ART line (artemisinin resistant) when compared with the NS parental line. The pymdr1 gene was mapped to a chromosome of 2.1 Mb in all lines analyzed. Reverse transcriptase–polymerase chain reaction and Western blot experiments confirmed the expression of the gene at the RNA and protein levels.