The tetraploid (2n = 32) Aconitum sect. Aconitum in the Eastern Carpathians, Southern Carpathians and Apušeni Mts. is represented by high-mountain A. bucovinese, A. firmum subsp. fissurae and their putative taxonomic hybrid A. ×nanum. The aim of the paper was to reveal which delimiting system: taxonomic vs. geographic-population better explains genetic variability (ISSR — Inter Simple Sequence Repeats) of the Aconitum individuals in the Eastern/Southern Carpathians. Twenty nine plants sampled in five populations within entire range of taxa distribution were assigned to genetic groups according to a Bayesian STRUCTURE analysis, neighbour-net classification (NN), and nonmetric multidimensional scaling ordination (NMDS). Three taxa were distributed in four (NN, NMDS) or two (STRUCTURE) genetic groups, and the partitioning of genetic variation with analysis of molecular variance (AMOVA) revealed the highest percentage of variation attributed to the four ISSR genetic groups (22.6%), then to the two STRUCTURE groups (18.9%) and three taxa (15.6%, all P < 0.001), and finally to the three geographic regions (6.5%, P = 0.013). Genetic groups harbored specimens from distant regions: A. f. subsp. fissurae had similar genetic profiles in the Southern Carpathians and Apušeni Mts. (100% support), and some specimens of A. bucovinense had genetic links with A. f. subsp. fissurae. The hybrid species A. ×nanum was genetically specific. We concluded that (i) genetic links between nowadays distantly located populations could have originated in the effect of ancient contacts and hybridization, (ii) probably in the Carpathians two ancient genetic centers of the A. sect. Aconitum existed and (iii) high genetic specificity of the hybrid species A. ×nanum deserves further studies.