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Ground squirrels are ecosystem engineers and keystone species in many open landscapes of Eurasia, America, and Africa. They are model objects for population studies, behavioural ecology, life-history theory, and conservation biology, the research areas where microsatellite analysis is widely applied and fruitful. So far, microsatellite markers have been developed for only few Palearctic ground squirrels. We tested and characterized 14 markers previously developed for ground squirrels and 10 new loci with tri-, tetra-, and five-nucleotide repeats in the yellow ground squirrel, Spermophilus fulvus, a species widely distributed in Eurasia and endangered in some regions. We found polymorphism in 10 loci, five of them were highly polymorphic (5–14 alleles). These markers will benefit studies of the population genetic structure, parentage, mating system, reproductive success, and interspecific hybridization as well as conservation efforts in S. fulvus and other close-related Eurasian ground squirrels. How to cite this article: Titov S.V., Batova O.N., Vasilieva N.A., Savinetskaya L.E., Tchabovsky A.V. 2020. Development and characterization of new polymorphic microsatellite markers for Eurasian ground squirrel Spermophilus fulvus (Lichtenstein, 1823) // Russian J. Theriol. Vol.19. No.2. P.131–135. doi: 10.15298/ rusjtheriol.19.2.03.