Turkish red pine (Pinus brutia) is a widespread and important forest tree species in Turkey, occurring mainly in southern, western, and northwestern Turkey, while the natural occurrence of Aleppo pine (Pinus halepensis) is restricted to 2 locations and is found sympatrically with Turkish red pine. In the present study sympatric populations of both species from Mugla and Adana provinces in Turkey were sampled, and the internal transcribed spacer 2 (ITS-2) region of ribosomal DNA was comparatively studied with sequence analysis. Analysis of molecular variance (AMOVA) demonstrated 100% of total molecular variation between the species in Mugla province, versus only 50.65% in Adana province. Construction of a phylogenetic tree with a bootstrap value of 92% revealed that Aleppo pine and Turkish red pine samples at the species level were well separated. Estimated F, values indicated that Turkish red pine and Aleppo pine were highly differentiated in Mugla province due to possible reproductive isolation, while the 2 species shared a more common genetic background due to possible natural hybridization in Adana province.