Twenty-one new disomic, polymorphic microsatellite DNA markers were isolated in Atlantic sturgeon, Acipenser oxyrinchus oxyrinchus. These markers yielded a total of 220 alleles in a survey of 16 fish; two to 21 alleles/locus were observed. Each locus segregated in a Mendelian fashion when tested in a family, and a set of 14 of the loci distinguished between siblings and half-siblings. Average observed heterozygosity ranged from 18.8 to 100.0%, and no linkage disequilibrium was detected. These loci should detect sufficient genetic diversity to allow kinship analysis for broodstock management, gene marking for stocking assessment and life history studies, and delineation of fine-scale population structure.