Trees are key components of historic gardens and are valuable sources of genetic diversity that need preservation to enrich native populations’ genetic basis in order to enhance long-term viability, and facilitate the species’ adaptation to changing climatic and environmental conditions. The primary objective of our study was to assess the genetic diversity, structure and relationships of the Tilia cordata Mill. allée at Széchenyi Castle in Nagycenk (UNESCO heritage site), Hungary, to infer the late-Baroque garden’s rich genetic heritage and conservation importance. The small leaved lime individuals at Nagycenk, with an age of 200 years, maintained high genetic diversity with non-significant inbreeding and high allelic richness and number of private alleles. Genetic diversity was similar to Central European natural populations and slightly higher compared to other plant collections, e.g. Eszterházy Castle garden at Fertőd. The surveyed trees exhibited two age groups unrelated with shallow genetic differentiation indicating the potential seed source origin from the local geographic area. Bayesian clustering of individuals showed significant differences from the individuals growing at Fertőd-Esterházy gardens (approx. in a 15 km distance) and other T. cordata cultivars. We concluded that the T. cordata collection of Nagycenk is a unique genetic heritage of the former natural vegetation due to its genetic variability and divergence, which would be a valuable source of future breeding programs and conservation.