Tree species with high ecological plasticity are recommended for use in urban green infrastructures. This study explores the genetic diversity of Tilia cordata, Tilia platyphyllos, and Tilia argentea, highlighting their contribution to urban landscapes. In this respect, the genetic variability of individuals from four populations of the Tilia genus has been analyzed using Inter Simple Sequence Repeats (ISSR) molecular markers, and some of their phenotypic characters were naked-eyed observed or determined by dendrometric measurements. Significant variability between the populations studied was determined. Cluster analysis identified two main groups: Tilia cordata I and Tilia cordata II populations formed the first cluster, and Tilia platyphyllos and Tilia argentea populations formed the second cluster. The results of the phenotypic analysis confirmed the genetic results separating the two populations of Tilia cordata from the populations of Tilia platyphyllos and Tilia argentea in relation to their visible morphological characters. Results obtained from dendrometric measurements analysis represent the foundation for further investigations on urban ecology. An essential potential practical use of the results: the establishment of more efficient ISSR primers to identify the Tilia species analyzed: UBC864, A12, UBC840, and A7 for Tilia cordata, A13 for Tilia platyphyllos and A7 for Tilia argentea has been demonstrated.