Cystoseira is a common brown algal genus widely distributed throughout the Atlantic and Mediterranean regions whose taxonomical assignment of specimens is often hampered by intra- and interspecific morphological variability. In this study, three mitochondrial regions, namely cytochrome oxidase subunit 1 (COI), 23S rDNA (23S), and 23S-tRNAVal intergenic spacer (mt-spacer) were used to analyse the phylogenetic relationships of 22 Cystoseira taxa (n = 93 samples). A total of 135 sequences (48 from COI, 43 from 23S and 44 from mt-spacer) were newly generated and analysed together with Cystoseira sequences (9 COI, 31 23S and 35 mt-spacer) from other authors. Phylogenetic analysis of these three markers identified 3 well-resolved clades and also corroborated the polyphyletic nature of the genus. The resolution of Cystoseira taxa within the three clades improves significantly when the inclusion of specimens of related genera was minimized. COI and mt-spacer markers resolved the phylogeny of some of the Cystoseira taxa, such as the C. baccata, C. foeniculacea and C. usneoides. Furthermore, trends between phylogeny, embryonic development and available chemotaxonomic classifications were identified, showing that phylogenetic, chemical and morphological data should be taken into account to study the evolutionary relationships among the algae currently classified as Cystoseira. The resolution of Cystoseira macroalgae into three well supported clades achieved here is relevant for a more accurate isolation and identification of natural compounds and the implementation of conservation measures for target species.