Mytilus mussels have been the object of much research given their sentinel role in coastal ecosystems and significant value as an aquaculture resource appreciated for both, its flavour and nutritional content. Some of the most-studied Mytilus species are M. edulis, M. galloprovincialis, M. chilensis and M. trossulus. As species identification based on morphological characteristics of Mytilus specimens is difficult, molecular markers are often used. Single-locus markers can give conflicting results when used independently; not all markers differentiate among all species, and the markers target genomic regions with different evolutionary histories. We evaluated the concordance between the PCR-RFLP markers most commonly-used for species identification in mussels within the Mytilus genus (Me15-16, ITS, mac-1, 16S rRNA and COI) when used alone (mono-locus approach) or together (multi-locus approach). In this study, multi-locus strategy outperformed the mono-locus methods, clearly identifying all four species and also showed similar specimen identification performance than a 49 SNPs panel. We hope that these findings will contribute to a better understanding of DNA marker-based analysis of Mytilus taxa. These results support the use of a multi-locus approach when studying this important marine resource, including research on food quality and safety, sustainable production and conservation.