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To determine the possible impact of over-exploitation on the genetic structure of Turkish red pine (Pinus brutia Ten.) populations, three natural and three over-exploited (human degraded) populations of the species in the Mediterranean region of Turkey were investigated with Randomly Amplified Polymorphic DNA (RAPD). With the 80 RAPD primers tested, 12 of them yielded 137 polymorphic RAPD fragments. Four of the studied populations maintained unique fragments. The mean proportion of polymorphic fragments for all populations ranged from 89.8 to 98.9% and there were no significant differences between natural (94.8%) vs. over-exploited populations (92.7%). The estimated heterozygosity values suggested that Turkish red pine maintains high levels of genetic diversity (range 0.24–0.28) though studied populations and grouped ones as natural (He = 0.28) vs. over-exploited (0.27) did not differ significantly. The mean FST value indicated that the large portion of the total genetic diversity was within populations (93%), but this value was lower in the natural populations (92%) than in the over-exploited ones (94%). In over-exploited populations, excess of homozygosity was observed (about 6% higher) as compared to natural populations, indicating impacts of inbreeding in P. brutia.