Aeshnidae Rambur, 1842 are impressive large insects distributed worldwide. Currently, over 500 species are recognized. Nevertheless, the phylogeny of this family is not completely understood. We applied molecular phylogenetic analysis using two popular phylogenetic markers, the mitochondrial COI gene fragment (barcoding sequence) and the nucleic ITS region, containing the ITS1, 5.8S rRNA, and ITS2 sequences. We used available and credible published sequences and 96 newly sequenced specimens. Our analysis involved all West Palaearctic species, all but one genera of the Holarctic Aeshnidae, and most genera worldwide, and is by far the largest molecular study of this family. The topology of all trees created with different algorithms and genes is in favour of the current taxonomic concept, with some remarkable outcomes. Aeshna Fabricius, 1775, was found to be diverged into several branches, especially with respect to the COI gene. Although it appeared not monophyletic in phylogenetic reconstructions based on the ITS region, the analysis of COI and joint analysis suggest its monophyly in the current taxonomical sense, with one notable exception. Aeshna isoceles (Müller, 1767) has fallen out of Aeshna in all analyses, so a new monophyletic genus, Isoaeschna gen. nov. is introduced for it. The genus Brachytron Evans, 1845 tightly clustered with Aeschnophlebia Selys, 1883, Epiaeschna Hagen in Selys, 1883, and Nasiaeschna Selys in Förster, 1900. Thus, we suggest subsuming these four genera under the priority name Brachytron. Tetracanthagyna Selys, 1883 clusters as expected with Brachytron in the ITS tree, but is an independent ancient clade of its own in all COI trees. The genus Polycanthagyna Fraser, 1933 syn. nov. is synonymised to Indaeschna Fraser, 1926. On the species level, we suggest that the American Aeshna septentrionalis Burmeister, 1839 be treated as a subspecies of A. caerulea (Ström, 1783), Aeshna caerulea septentrionalis. We synonymize Gynacantha hyalina Selys, 1882 with Gynacantha subinterrupta Rambur, 1842. Our analysis provides new insights on the tight relationships of the circumboreal species Aeshna juncea and A. subarctica and the intraspecies phylogeny of Aeshna juncea.