In Rwanda, frequent mutations in the pfdhfr and pfdhps genes of Plasmodium falciparum have suggested intense sulfadoxine-pyrimethamine resistance. However, data on pfmdr1 are not available but might be important in the context of the first-line treatment with artemether-lumefantrine. During a survey among 749 children under five years of age in southern highland Rwanda, 104 P. falciparum isolates were obtained. Parasite polymorphisms associated with drug sensitivity were typed including the genes pfdhfr, pfdhps, pfmdr1, and pfcrt. Plasma concentrations of chloroquine and pyrimethamine were measured by ELISA. Treatment with artemether-lumefantrine within the preceding two weeks was stated by 12.5% of the respondents; chloroquine in plasma was detected in 17.6%, pyrimethamine in none. Isolates with pfdhfr triple and pfdhps double/triple mutations occurred in 75% and 93%, respectively; 69% of the isolates comprised pfdhfr/pfdhps quintuple or sextuple mutations associated with high-grade sulfadoxine-pyrimethamine resistance. Pfdhfr L164 was absent. The pfmdr1 pattern revealed more than 50% of the F184 polymorphism and almost 40% of the N86-F184-D1246 allele combination known to be selected in infections reappearing following artemether-lumefantrine treatment. Molecular markers demonstrate intense antifolate drug resistance of P. falciparum in southern Rwanda. The present, first-time data on pfmdr1 alleles from Rwanda reveal a pattern which might reflect a predominance of wild types for some alleles or, alternatively, substantial artemether-lumefantrine pressure on the local parasite population.