In an in vitro study of P. falciparum field isolates, cross-resistance existed between pyrimethamine and chlorcycloguanil and the triple mutant increased resistance to the drugs 225-fold and 48- fold, PF-04217903 respectively. While in vitro the presence of this triple DHFR mutant at codons 108, 51, and 59 increased drug resistance to both antifolates, this combination of mutations was only associated with clinical treatment failure with sulfadoxine-pyrimethamine but not with chlorproguanil-dapsone. The addition of the I164L mutation results in clinical failure of chlorproguanil-based therapies. In 1968 a study of the parasite P. berghei in mice showed that there is some cross-resistance between parasites exposed to chlorcycloguanil and PF-2341066 pyrimethamine, with exposure to chlorcycloguanil inducing broader resistance than pyrimethamine. It was clear then that certain resistant strains may be countered by using a different drug targeting the same mutant enzyme, but that multiply resistant lines may also arise. Tracing the mutational trajectories of those two drugs now affords an evolutionary explanation for those results from many decades ago: The shape of the underlying adaptive landscapes of the drugs in question affects the genotypes that will emerge—even between drugs targeting the same enzyme in the same manner. Drug resistance is a major factor in determining the course of treatment for malaria and other infectious diseases. The ability to not only pinpoint mutations that lead to drug resistant phenotypes, but also to predict the route evolution is most likely to take once drug pressure is exerted, are invaluable assets in preserving the efficacy of chemotherapy. Knowledge regarding the progression of evolutionary trajectories can be determined in an in vitro system in a relatively short period of time as compared to tracking trends in the field. Scientists and clinicians may then have advance notice of the mutational progression in trajectories that are likely to lead to the exhibition of high drug resistance allowing them to use their limited resources in a more focused way as they survey for the emergence of drug resistance. Transgenic systems such as the one used in this study cannot address all the complexities involved in the evolution of drug resistance in clinical settings.