In this study, we isolated 21 novel polymorphic microsatellite DNA loci from the pen shell Atrina pectinata using magnetic-bead hybridization enrichment. The characteristics of these loci were tested using a population of 30 individuals collected from the Penglai coast, Shandong Province. The number of alleles ranged from 2 to 13, and polymorphism information content (PIC) varied from 0.1730 to 0.8954. Values for observed heterozygosity (HO) and expected heterozygosity (HE) ranged from 0.0714 to 0.9231 and from 0.1948 to 0.9237, respectively. Four loci deviated significantly from Hardy-Weinberg equilibrium. The newly developed microsatellite markers will be beneficial in assessing the genetic diversity, population structure and genetic conservation of A. pectinata, and in other relevant research.