Species of the genus Cystoseira are particularly hard to discriminate, due to the complexity of their morphology, which can be influenced by their phenological state and ecological parameters. Our study emphasized on the relevance of two kinds of analytical tools, (1) LC/ESI-MSn and (2) 1H HR-MAS NMR, also called in vivo NMR, to identify Cystoseira specimens at the specific level and discuss their taxonomy. For these analyses, samples were collected at several locations in Brittany (France), where Cystoseira baccata, C. foeniculacea, C. humilis, C. nodicaulis and C. tamariscifolia were previously reported. To validate our chemical procedure, the sequence of the ITS2 has been obtained for each species to investigate their phylogenetic relationships at a molecular level. Our study highlighted the consistency of the two physico-chemical methods, compared to "classical" molecular approach, in studying taxonomy within the genus Cystoseira. Especially, LC/ESI-MSn and phylogenetic analyses converged into the discrimination of two taxonomical groups among the 5 species. The occurrence of some specific signals in the 1H HR-MAS NMR spectra and/or some characteristic chemical compounds during LC/ESI-MSn analysis could be regarded as discriminating factors. LC/ESI-MSn and 1H HR-MAS NMR turned out to be two relevant and innovative techniques to discriminate taxonomically this complex genus.