In Plasmodium falciparum, resistance to folate inhibitors like pyrimethamine is mediated by point mutations in the target gene dihydrofolate reductase (dhfr). The resistance to pyrimethamine increases with the accumulation of particular point mutations. These mutations also confer increased resistance to chlorcycloguanil, the active metabolite of chlorproguanil and one component of a newly introduced DHFR inhibitor, LapDap. One genotype (16V/108T) has been previously identified that confers resistance to cycloguanil but not to pyrimethamine. This study was designed to identify novel alleles that might confer resistance to chlorcycloguanil, but escape the surveillance methods currently in place for common pyrimethamine-resistant alleles. Directed mutagenesis was performed using the wild type and the common pyrimethamine-resistant allele, 51I/59R/108N, to determine the effect of the 16V and 108T mutations on enzyme activity and drug resistance. In addition, we randomly mutagenized the 51I/59R/108N allele and identified nine novel alleles that could confer resistance to chlorcycloguanil. These yeast strains were also resistant to pyrimethamine, but retained sensitivity to the experimental DHFR inhibitor, WR99210. None of the alleles generated in this study was as resistant to chlorcycloguanil as the common quadruple mutant, 51I/59R/108N/164L. In addition, selection of high levels of chlorcycloguanil resistance in parasites that carry the 51I/59R/108N allele will require two directed steps, a change from 108N to 108T followed by a mutation from A16 to 16V. The resulting allele, 16V/51I/59R/108T is highly resistant to chlorcycloguanil, but 200-fold more sensitive to pyrimethamine than the 51I/59R/108N allele.