Reintroductions play a key role in conservation biology. Their long-term success strongly depends on maximizing the genetic diversity of founders, as well as on ensuring ecological connectivity with adjacent populations. Accordingly, evaluating the conservation status of reintroduced populations to limit the loss of their genetic diversity requires long-term genetic monitoring. The Griffon Vulture population of central Italy originated from 93 birds sourced from wildlife rehabilitation centers in Spain from 1994 to 2002, without prior genetic characterization. To conduct a post-release genetic assessment, feather samples from 155 free-living individuals, collected from 2013 to 2018, were analyzed at biparental Short Tandem Repeat loci (STRs). Genetic diversity parameters, mean number of alleles (A), mean effective number of alleles (Ae), expected heterozygosity (He), and observed heterozygosity (Ho), were estimated and compared with those reported for native and reintroduced European Griffon Vulture populations. Individual inbreeding coefficient (FIS) and effective population size (Ne) were also assessed. Genetic variability (A=7.8; Ae=3.69; He=0.64; Ho=0.63) and effective population size (Ne ≈125) are consistent with medium-term viability. Furthermore, field observations and genetic data suggest connectivity with other populations, including those in Spain, France and the Balkans, supporting the potential role of this population as a genetic bridge between western and eastern populations. This study represents the first population-level genetic assessment based on nuclear markers of the reintroduced continental Griffon Vulture population in Italy, providing a baseline for future genetic monitoring and assessments of gene flow and connectivity among European populations.