The European wildcat Felis silvestris silvestris is one of the most endangered mammals in Poland. In 1954, when the Tatra National Park (TNP) was established in the highest part of the Polish Carpathians, the wildcat was considered a rare species but nevertheless present in this region. However, its occurrence was never properly recorded there. The aim of this study was to verify whether wildcats occur in the TNP using genetic methods to distinguish wildcats from domestic cats F. s. catus. Between March and May 2015, hair samples were collected from 40 lure sticks treated with valerian, a cat attractant, in a region of the park with presumed high habitat suitability for wildcats. Neither wildcats nor hybrids with domestic cats were identified using control region haplotype sequencing and analysis of different nuclear markers. However, thirteen samples indicating the presence of the domestic cat in the protected area were collected. The Bayesian clustering analyses of microsatellite and SNP genotypes revealed no evidence for any admixture with wildcats. While our study cannot prove the absence of wildcats in the study area, it strongly suggests that wildcats are at least very rare in the region and a continued rigorous monitoring is recommended.