The resistance of Plasmodium falciparum to sulfadoxine-pyrimethamine (SP) is an emerging public health threat. Resistance to these drugs is associated with point mutations in the genes encoding dihydropteroate synthase (DHPS) and dihydrofolate reductase (DHFR). We describe here an assay using real-time PCR and sequence-specific probes that detects these mutations. Using DNA from plasmids, cultured strains, and clinical samples, real-time PCR could distinguish four DHPS polymorphisms (codons 437, 540, 581, and 613) and three DHFR polymorphisms (codons 51, 59, and 108). This assay is rapid and sensitive, with a detection limit of 10 copies in most cases. This assay is amenable to large-scale studies of drug resistance.