ABSTRACT The anchovy, Engraulis ringens, is an economically valuable fish, and the most heavily exploited resource in the Humboldt Current System. To analyze its genetic structure and diversity, microsatellite markers were developed using 454 pyrosequencing. A total of 27,352 reads containing di-, tri-, tetra-, penta-, and hexanucleotide microsatellite repeat units were identified from 136,537 reads. Among 80 loci containing more than six repeat motifs, 32 primer sets (40%) produced reproducible PCR products, and all of these loci were polymorphic. Some loci showed deviations from HWE and possible null allele's presence, results of an excess of homozygotes. In an analysis of 45 individuals from one E. ringens population, the number of alleles per locus ranged from 2 to 33, observed heterozygosity ranged from 0.171 to 0.976, and the probability of identity values ranged from 0.006 to 0.513. These microsatellites will be useful for numerous ecological studies focused on this important pelagic fish; including the examination of population genetic structure, estimating effective population size and providing information for fisheries management.