Pulsatilla x hakelii Pohl. is the most common hybrid between Pulsatilla patens and Pulsatilla pratensis. It occurs in the natural environment, most often in sites together with parental forms. Pulsatilla × hackelii is morphologically intermediate between the pure Pulsatilla species. Separation of hybrid individuals from pure Pulsatilla species requires a whole set of morphological features. Completed plastid genome provide molecular evidence for the hybrid origin of Pulsatilla × hackelii. In our study we presents the – genotypic characteristics of Pulsatilla x hakelii based on microsatellites markers developed for Pulsatilla vulgaris. A specimen of the hybrid was found in the Świętokrzyskie Mountains, near Bocheniec village, in the vicinity of a forest road in a fresh mixed coniferous forest (Querco roboris-Pinetum (W.Mat. 1981) J.Mat. 1988). We amplified a set of 10 microsatellites in two multiplex PCR reactions. From the 10 analyzed loci, we successfully amplified eight. The analyzed microsatellites in population studies of three Pulsatilla species showed some species specific alleles. In case of the Pulsatilla x hakelii sample, we obtained PCR products in the case of 5 loci developed for P. vulgaris, but successfully crossamplified in P. patens and P. pratensis. Additionaly we obtained results for 2 loci, which crossamplified only for Pulsatilla patens and 1 only for Pulsatilla pratensis. Among the investigated markers, 2 were monomorphic and the remaining six were polymorphic, what making them highly informative in population studies for pasque-flower species as Pulsatilla patens, P. pratensis and P. vernalis. The obtained results indicated that analized microsatellites markers can be used for hybrids identification, although there is a need for further research on a larger number of hybridized individuals.