The RDA Genebank at the National Agrobiodiversity Center (NAAS, RDA, Republic of Korea) has conserved about 182,000 accessions in 1777 species and is working at preserving agricultural genetic resources for the conservation and sustainable utilization of genetic diversity. The detection of genetic variability in conserved resources is important for germplasm management, but the molecular evaluation tools providing genetic information are insufficient for underutilized crops, unlike those for major crops. In this regard, the Korean National Agrobiodiversity Center has been developing microsatellite markers for several underutilized crops. We designed 3640 primer pairs flanking simple sequence repeat (SSR) motifs for 6310 SSR clones in 21 crop species. Polymorphic loci were revealed in each species (7–36), and the mean ratio of polymorphic loci to all the loci tested was 12%. The average allele number was 5.1 (2.8–10.3) and the expected heterozygosity 0.51 (0.31–0.74). Some SSRs were transferable to closely related species, such as within the genera Fagopyrum and Allium. These SSR markers might be used for studying the genetic diversity of conserved underutilized crops.