Malaria parasites break down host haemoglobin into peptides and amino acids in the digestive vacuole for export to the parasite cytoplasm for growth: interrupting this process is central to the mode of action of several antimalarial drugs. Mutations in the chloroquine (CQ) resistance transporter,pfcrt, located in the digestive vacuole membrane, confer CQ resistance inPlasmodium falciparum, and typically also affect parasite fitness. However, the role of other parasite loci in the evolution of CQ resistance is unclear. Here we use a combination of population genomics, genetic crosses and gene editing to demonstrate that a second vacuolar transporter plays a key role in both resistance and compensatory evolution. Longitudinal genomic analyses of the Gambian parasites revealed temporal signatures of selection on a putative amino acid transporter (pfaat1)variant S258L, which increased from 0% to 97% in frequency between 1984 and 2014 in parallel with thepfcrt1K76Tvariant. Parasite genetic crosses then identified a chromosome 6 quantitative trait locus containingpfaat1that is selected by CQ treatment. Gene editing demonstrated thatpfaat1S258Lpotentiates CQ resistance but at a cost of reduced fitness, whilepfaat1F313S, a common southeast Asian polymorphism, reduces CQ resistance while restoring fitness. Our analyses reveal hidden complexity in CQ resistance evolution, suggesting thatpfaat1may underlie regional differences in the dynamics of resistance evolution, and modulate parasite resistance or fitness by manipulating the balance between both amino acid and drug transport.