Existing parentage analyses of large raptors have mostly used naturally shed feathers as a source of DNA. There is evidence that eagle nests are sometimes visited by conspecifics. Are these visitors involved in extra pair-paternity? More than 400 feather samples of nestlings and full-grown White-tailed Eagles were obtained in May at 13 nest sites in the Czech Republic, mainly during the years 2010–2016. The samples were genotyped at six microsatellite loci to determine the identity and number of individuals occurring at each nest site and to test genetic monogamy. In the end, 110 unique genotypes of possible parents were identified from moulted feathers found below nests and were used in a parentage analysis together with 86 offspring genotypes. Interestingly, more than two adult genotypes were sampled from shed feathers in one-third of 64 checks of 11 nests, but nest success was not affected by the number of genotypes. No case of extra-pair paternity was recorded. The results showed that feathers of individuals other than the parent pair can be found below nests of White-tailed Eagles during a single breeding season, and the identity of parents at individual nests can change even during seven years of study, which is inconsistent with the expected longevity, lifelong monogamy and presumed territoriality of White-tailed Eagles. We cannot recommend DNA sampling of nesting pairs on the basis of feathers found below eyries, because the most likely parent was recorded in the appropriate year only in 58% of cases with this method.