Genetic variability and differentiation of six populations of Tilia rubra from Hyrcanian forests of the north of Iran were analyzed using random ampliﬁed polymorphic DNA (RAPD) markers. Seven primers pairs used to study six populations, in six localities located in at different altitude (250–2500 m a.s.l) but within the distance 50 km, provided 70 reliable bands, of which 29 (41.4%) were polymorphic. The average population diversity using the Shannon’s index (I) was 0.22 and ranged from 0.22 for Bandbon population to 0.08 for the Salahedinkola population. The mean heterozygosity varied from 0.013 to 0.06 within the populations of T. rubra. The AMOVA analysis showed a significant genetic differentiation within populations and 97% of the total variation occurred within populations and only 3% occurred among populations. Cluster analysis using the UPGMA method grouped all individuals into four clusters. The results did not correspond with the populations origin. The low inter-population differentiation and the lack of significant correlation between geographic and genetic distances suggest intensive gene flow among populations of Tilia rubra.