Microsatellite-containing regions were isolated and characterized in Anopheles (Nyssorhynchus) marajoara, a primary vector of malaria parasites in northeastern Amazonia, Brazil. An enrichment protocol yielded 500 positive clones. We designed primers to amplify 40 unique microsatellites, 11 of which amplified cleanly and were polymorphic. A survey of 323 individuals showed that these loci are highly variable with the number of alleles ranging from 11 to 52, and expected heterozygosity ranging from 0.64 to 0.95. These markers will be useful for studies of population structure and intraspecific variation in A. marajoara.