Membrane-associated ATP binding cassette (ABC) transport proteins hydrolyze ATP in order to translocate a broad spectrum of substrates, from single ions to macromolecules across membranes. In humans, members from this transport family have been linked to drug resistance phenotypes, e.g., tumour resistance by enhanced export of chemotherapeutic agents from cancer cells due to gene amplifications or polymorphisms in multidrug resistance (MDR) protein 1. Similar mechanisms have linked the Plasmodium falciparum PfMDR1 transporter to anti-malarial drug resistance acquisition. In this study, the possible involvement of two related MDR proteins, PfMDR2 and PfMDR5, to emerging drug resistance is investigated by a reverse genetics approach. A homologous double crossover strategy was used to generate P. falciparum parasites lacking the Pfmdr2 (PfΔmdr2) or Pfmdr5 (PfΔmdr5) gene. Plasmodium lactate dehydrogenase activity was used as read-out for sensitivity to artemisinin (ART), atovaquone (ATO), dihydroartemisinin (DHA), chloroquine (CQ), lumefantrine (LUM), mefloquine (MQ), and quinine (QN). Differences in half maximal inhibitory concentration (IC₅₀) values between wild type and each mutant line were determined using a paired t-test. Both PfΔmdr2 and PfΔmdr5 clones were capable of asexual multiplication. Upon drug exposure, PfΔmdr2 showed a marginally decreased sensitivity to ATO (IC₅₀ of 1.2 nM to 1.8 nM), MQ (124 nM to 185 nM) and QN (40 nM to 70 nM), as compared to wild type (NF54) parasites. On the other hand, PfΔmdr5 showed slightly increased sensitivity to ART (IC₅₀ of 26 nM to 19 nM). Both Pfmdr2 and Pfmdr5 are dispensable for blood stage development while the deletion lines show altered sensitivity profiles to commonly used anti-malarial drugs. The findings show for the first time that next to PfMDR2, the PfMDR5 transport protein could play a role in emerging drug resistance.