<p>The ecological significance of mixed forests, featuring an increase of broadleaf tree species such as large-leaved lime (<i>Tilia platyphyllos</i> Scop.), is widely acknowledged in Europe. Large-leaved lime holds importance within traditional landscapes and has high cultural value in open landscapes, urban areas, and recreational forests. In response to climate change, the forestry sector seeks to enhance forest structure by establishing mixed stands, thereby increasing the demand for appropriate forest reproductive material (FRM). However, until now, there has been limited knowledge regarding the genetic structure and diversity of large-leaved lime populations in Bavaria, Southern Germany. This study aims to evaluate genetic structure and diversity while selecting potential seed stands and gene conservation units (GCU) for large-leaved lime. We analysed 15 natural populations and two seed orchards in Bavaria, using 11 variable microsatellite loci. The results of AMOVA show that genetic variation is largely within populations (89%), with 6% between populations (F_ST = 0.06) and 5% among individuals. Bayesian clustering identified two genetic clusters: one (K1) in northern Bavaria and another (K2) in southern Bavaria. Three populations in central Bavaria showed a relatively balanced admixture of both clusters, indicating a transitional zone between the northern and southern groups. This study is the first to provide comprehensive genetic data on <i>T. platyphyllos</i> in Bavaria, offering new insights into its genetic diversity and structure. The findings support the sustainable use and conservation of large-leaved lime, providing a foundation for informed selection of GCUs and seed stands in Bavaria.</p>