Premise Gladiolus palustris (Iridaceae) is an endangered European perennial tetraploid herb with special conservation interest in the European Union. Microsatellite markers can serve as effective tools for the conservation genetics of this species. Methods and Results We utilized a 454 pyrosequencing approach to identify simple sequence repeat (SSR) regions in a microsatellite‐enriched library. Of all SSR regions, 46 were screened for specific PCR amplification, and 15 were found to be applicable in the target species. We found 1.62–3.08 alleles per population (effective alleles: 1.58–2.08) that indicated moderate to high genetic diversity values (0.28–0.44) in three pilot populations. Cross‐species amplification was less effective in G. imbricatus and G. tenuis. Conclusions The primers reported here can be used for the population genetic characterization of G. palustris. They will help us to better understand the conservation genetics of this highly endangered species.