Aim of study: To characterize and compare the genetic resources and gain some insights into the evolutionary history of Aleppo pine (Pinus halepensis Mill.) and Brutia pine (Pinus brutia Ten.) species which are both distributed across more than 8 million hectares of area in the Mediterranean Basin. Area of study: Fifty-six populations from eight Mediterranean basin countries where P. halepensis and P. brutia species are located. Materials and methods: We analyzed 1344 seeds belonging to 56 populations using five cpSSR primers (Pt15169, Pt30204, Pt41093, Pt87268, and Pt110048). Main results: The analysis of molecular variance (AMOVA) revealed that the genetic diversity among the Brutia pine populations was slightly higher than that of Aleppo pine (27.06% and 24.27%, respectively). The Aleppo pine populations separately displayed a clear east-west differentiation across the Mediterranean Basin, confirming previous results using other markers. Although the Brutia pine populations showed no spatial genetic pattern, geographically close populations and/or populations from their continual distribution range were genetically closer than the fragmented and/or ecologically marginal populations. Research highlights: The seven Aleppo pine populations from the eastern range (Türkiye, Greece, and Italy) were more than twofold diverse than the 13 populations from the western range (Spain and Morocco). The eastern range of Aleppo pine and Brutia pine populations had similar levels of genetic diversity parameters. These results suggested that the Eastern Mediterranean Basin is a possible genetic diversity center for the two pine species.