This is the first report on the composition and variability of the needle-wax n-alkanes in natural populations of Pinus nigra in Serbia. Samples of 195 trees from seven populations belonging to several infraspecific taxa (ssp. nigra, var. gocensis, ssp. pallasiana, and var. banatica) were analyzed. In general, the size of the n-alkanes ranged from C16 to C33, with the exception of ssp. nigra, for which it ranged from C18 to C33. The most abundant were C23-, C25-, C27-, and C29-alkanes. The needle waxes of Populations I–III and V were characterized by a higher content of C23-, C25-, and C27-alkanes and a lower content of C24-, C26-, C28-, and C30-alkanes, compared to the other populations, and the trees of these populations could be assigned to ssp. nigra. The samples of Population VI were characterized by higher amounts of C22-, C24-, C30-, and C32-alkanes and lower amounts of C25- and C27-alkanes, and the trees could be considered as ssp. pallasiana. The samples of Population VII, consisting of trees belonging to var. banatica, were richer in C29-, C31-, and C33-alkanes. The wax compositions of Populations IV and V, both composed of trees previously determined as P. nigra var. gocensis, showed a tendency of splitting. Indeed, the alkane composition of Population IV was closer to that of ssp. pallasiana pines, while that of Population V was more similar to that of ssp. nigra pines. From the results presented here, it is obvious that in the central part of the Balkan Peninsula, significant diversification and differentiation of the populations of black pine exists, and these populations could be defined as different intraspecific taxa. Our results also indicate the validity of n-alkanes as chemotaxonomic characters within this aggregate.