Key message This study identified 10 fir populations that may be hybridogenous. The immediate activities on conservation of these polymorphic fir populations should be supported as hybrids may be ecologically and economically superior to the parent species. This is the first report on morpho-anatomical differentiation of both lower-rank and upper-rank needles in Abies alba , A. x borisii-regis, and A. cephalonica , using 18 native populations from almost the entire assumed hybrid zone in the Balkan Peninsula. The 35 morpho-anatomical characteristics were analyzed biometrically, and 18 with the application of multivariate statistical analysis. Clinal pattern in distribution of morpho-anatomical variation over the broader transition zone between the putative parent species was observed. Furthermore, the results of the biometric analyses justify the validity of this method for differentiating between the eastern A. alba lineage and A. cephalonica , for both needle types. Thus, the results of this study show clear differences between A. alba and A. cephalonica , but unstable characteristics of A. x borisii-regis . This finding supports the hypothesis that A. x borisii-regis is an unstabilized taxon produced by recent hybridization and introgression between A. alba and A. cephalonica . Based on the multivariate statistical analysis, 10 of the studied fir populations may be hybridogenous within a wider geographical area (southwestern and southern Bulgaria, southernmost North Macedonia and almost the entire Greece). The obtained results did not indicate a hybrid status for two fir populations from Peloponnese and one from the Cephalonia Island (pure A. cephalonica populations), which partly agrees with the recent molecular data.