This is the first report on morpho-anatomical and phytochemical differentiation of 19 native populations representing different Pinus nigra J.F.Arnold subspecies (banatica (Borbás) Novák, dalmatica (Vis.) Franco, nigra, and pallasiana (Lamb.) Holmboe) in the Balkans and Southern Carpathians. The 9 morpho-anatomical characteristics and 10 headspace volatiles of needles were analyzed with multivariate statistical analyses. The combination of results from all multivariate analyses with both types of markers revealed that P. nigra is differentiated into three groups within the studied area (the Dalmatian coast, Greece, and the rest of the Balkans with the Southern Carpathians). The first group included the population from an island in Dalmatia that corresponds to P. nigra subsp. dalmatica. The third group consisted of populations from continental Croatia, Bosnia and Herzegovina, Serbia, Romania, and Bulgaria, which corresponds to P. nigra subsp. nigra. In light of the recent molecular data that indicated that the Greek populations (the second group) represent a distinct genetic lineage of P. nigra placed between the populations from the principal area (P. nigra subsp. nigra) and Turkey (P. nigra subsp. pallasiana), one can speculate that there is one more subspecies of P. nigra in this region that corresponds to populations from Greece. Extending our analyses to Asia Minor and Crimea could bring additional results that would be valuable for clarifying the intriguing issue of the diversification of P. nigra in the eastern part of its range.