After a major increase in incidence between the 1970s and the 1990s, the Brazilian Amazon region now accounts for the most cases of Plasmodium falciparum malaria in the Americas. Polymorphism of 10 microsatellite loci in the P. falciparum genome was studied in 196 isolates obtained from 5 populations in the region. There was significant multilocus linkage disequilibrium, particularly within populations with lower proportions of mixed-genotype infections. However, most multilocus genotypes in different isolates were distinct, and there was no evidence of any recent epidemic expansion of particular clones. Genetic divergence between populations was very substantial but did not fit a simple model of isolation by distance. Thus, different foci of P. falciparum in Brazil are quite independent, with distinct population structures and minimal gene flow, a finding that has implications for strategies to control infection and to contain the spread of drug resistance at a regional level.