Using a range of laboratory-adapted and genetically modified Plasmodium falciparum parasite isolates, we investigated the interaction between dihydroartemisinin and piperaquine (PIP), the individual components of an artemisinin combination therapy currently under development, in addition to the role of known drug resistance genes in parasite susceptibility in vitro. All but one parasite line investigated displayed an interaction of dihydroartemisinin and PIP that was antagonistic, although the degree of antagonism was isolate dependent. In terms of resistance markers, the pfcrt haplotypes CVIET and SVMNT were positively associated with reduced sensitivity to PIP, with parasites carrying the South American CQR (SVMNT) allele being generally less sensitive than CVIET parasites. Parasites carrying the CQS (CVMNK) allele displayed a further increase in PIP sensitivity compared with CVIET and SVMNT parasites. Our data indicate that PIP sensitivity was not affected by pfmdr1 sequence status, despite positive correlations between the structurally related compound amodiaquine and pfmdr1 mutations in other studies. In contrast, neither the pfcrt nor pfmdr1 sequence status had any significant impact on susceptibility to dihydroartemisinin.