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In January 2007, Tanzania replaced sulfadoxine-pyrimethamine (SP) with artemether-lumefantrine for treatment of uncomplicated malaria. This study examined the impact of widespread SP use on molecular markers of Plasmodium falciparum drug resistance in blood samples from persons living in two villages in Korogwe District, Tanzania, from 2003 through 2007. The prevalence of the P. falciparum dihydropteroate synthase (Pfdhps) gene 581G mutation increased from 12% in 2003 to 56% in 2007 (P < 0.001), resulting in an increase in the triple mutant Pfdhps haplotype SGEGA from 8% to 32% (P < 0.001). In contrast, the chloroquine-sensitive P. falciparum chloroquine resistance transporter (Pfcrt) CVMNK haplotype increased from 6% to 30% (P < 0.001). The dramatic increase of the triple Pfdhps mutant SGEGA haplotype may endanger the continued use of SP for intermittent presumptive treatment of pregnant women (IPTp). Further studies are needed to determine the importance of Pfdhps SGEGA haplotype parasites on the efficacy of SP for IPTp.