New nuclear microsatellite markers (SSRs) were developed for Aesculus hippocastanum, a relict tree species from the Balkan Peninsula. The development of microsatellites was done using the Illumina MiSeq PE300 platform. Out of a set of 500 SSRs designed, a subset of 13 loci was tested using 290 individ­uals from seven natural populations. Twelve species-specific loci were polymorphic. The number of alleles per locus ranged from 2 to 17 and expected heterozygosity from 0.089 to 0.800 with a mean value of 0.484. The population of Kalampaka had the lowest value of allelic richness (2.63) and gene diversity in compari­son to the remaining populations. STRUCTURE analysis confirmed isolation of population Mariolata from the southern edge of the species range and genetic similarity among populations from the Pindos Mts. Ad­ditionally, the utility of new SSRs in 29 individuals from nine other Aesculus taxa was tested. Eleven markers gave polymorphic products for all tested species. For 24 individuals, a high-quality product was obtained for each marker. Results confirmed the utility of specific markers for future population genetics studies.