The state of Bolivar in Venezuela experiences episodic outbreaks of multidrug-resistant Plasmodium falciparum malaria. We obtained P. falciparum-infected blood samples in Bolivar in 1998-2000, and performed molecular assays for mutations conferring resistance to the antifolate combination of sulfadoxine-pyrimethamine (SP) and to chloroquine. All infections carried the dihydrofolate reductase (dhfr) S108A and N51I mutations, and 45% of the infections had the dhfr C50R mutation, which has been implicated in mid-level resistance to SP. Two dihydropteroate synthase (dhps) mutations also involved in SP resistance, A581G and K540E, were detected in 90% and 67% of the samples, respectively. The dhfr 1164L mutation, which confers high-level resistance, was not identified. The P. falciparum chloroquine resistance transporter (pfcrt) K76T mutation, which is critical for chloroquine resistance, was found in 167 of 168 infections. Six dhfr/dhps allelotypes and four pfcrt-resistant alleles were observed. Their interrelationships suggest a semi-clonal propagation of P. falciparum malaria in Bolivar, and an invasion of multi-resistant pathogens from Brazil. Despite national restrictions on the use of SP and chloroquine, genotypic resistance to these therapies remains widespread in Bolivar.