Silverleaf nightshade (2n=2x=24) is a serious weed of the family Solanaceae, for which no specific markers are available to date. In order to investigate the extent and distribution of genetic diversity among accessions of silverleaf nightshade, we developed twenty-three simple sequence repeats (SSR) markers from publicly available nucleotide and EST databases for silverleaf nightshade. Eleven of them were single-locus polymorphic markers. The number of alleles among these loci ranged from 2 to 4. The observed and expected heterozygosity ranged from 0 to 0.97 and 0.07 to 0.64, respectively. Fourteen SSR markers enabled to amplify alleles in morphologically similar species quena. These results proved that the SSR markers that we developed could be useful for (i) determining genetic diversity and structure among natural populations of silverleaf nightshade and (ii) identification of silverleaf nightshade and quena ecotypes. This is the first set of species-specific SSR markers identified in silverleaf nightshade, which could contribute to the better understanding of genetic diversity of silverleaf nightshade and related species.