Markers of Plasmodium falciparum resistance to chloroquine (CQ) and pyrimethamine-sulfadoxine (PYR-SDX) are widespread in areas where malaria is endemic. In an area where the use PYR-SDX is negligible, the Ashanti Region of Ghana, West Africa, adult individuals were enrolled in an analysis of CQ- and PYR-SDX-associated molecular resistance markers in 2001 (n = 177) and 2003 (n = 180). Parasite prevalence, as assessed by PCR assays, were 56.5 and 48.8% in 2001 and 2003, respectively. A high frequency of CQ, PYR, and SDX resistance markers was observed, whereby, as a weak trend, the frequency was higher in 2003. The quintuple combination of three pfdhfr mutations and two pfdhps mutations has previously been recognized to be the most important determinant of PYR-SDX resistance. Approximately 60% of parasite carriers harbored fourfold mutated parasites, indicative of a considerable risk for a switch to high-level PYR-SDX resistance in an area where the rate of PYR-SDX use is low. Among the factors contributing to the high frequency of PYR-SDX resistance-associated mutations are background use of PYR-SDX, past use of PYR for malaria prophylaxis, cross-resistance of trimethoprim with PYR, and the sufficient biological fitness of resistant parasites in the absence of drug pressure.