The southern part of Europe is one of the most species-rich regions from the point of view of the genus and subgenus Ixodes. However, numerous unresolved or questionably interpreted issues exist in the context of tick species indigenous to Mediterranean countries, such as the validity of Ixodes festai, synonymy of Ixodes tatei with Ixodes eldaricus (never tested molecularly) or the haplotype heterogeneity of Ixodes gibbosus. In this study, 21 specimens of six tick species from the subgenus Ixodes were compared morphologically with high resolution digital microscopy and also analyzed with molecular-phylogenetic methods based on two mitochondrial genetic markers. The nymphs of I. eldaricus and I. tatei showed differences in the morphology of the scutum and basis capituli. Both the nymph and the females of I. festai could be distinguished from those of I. eldaricus, I. ventalloi and I. acuminatus. A female tick resembled I. gibbosus but was also different from this species, based on its descriptions. Analysis of phylogenetic relationships confirmed with moderate to strong support that all six species examined in this study represent different taxa of the subgenus Ixodes, including a previously unknown sister species to I. gibbosus. The latter is recognized and described here as a new species, Ixodes paragibbosus Hornok and Kontschán, sp. nov. Based on findings of this study, the tick species I. tatei Arthur, 1959 should be resurrected and reestablished. Morphological and phylogenetic comparisons performed here (including the first barcoding sequences of I. eldaricus and I. festai) confirm that the latter is a valid species, distinct from both I. eldaricus and I. ventalloi. For the differential diagnosis of the above species, the results highlight the importance of observing (among other structures) the auriculae, the internal spur of coxa I and the hypostome.