Several strategies carried out for the application of proteomic methodologies to species and populations are summarized. Species of the genus Mytilus are used as a model organism to explain these strategies. The proteomics needed to differentiate populations, species, following some different approaches are provided. Moreover, there is an explanation of when it is most critical to carry out a rigorous analysis of this type. Sample preparation, selection of the most appropriate tissue of this organism for a comparative analysis, two-dimensional gels, computer analysis, mass spectrometry (MS) combined with two-dimensional electrophoresis (2-DE) are described. Then, the inconveniences of working with species like those of the genus Mytilus that are poorly represented in databases will be presented along with a description of how to approach this problem. Likewise, the review will include the strategy to follow when dealing with species, like mussels, that present a high degree of genetic polymorphism. The different protein-expression-based strategies used to approach the problem of differentiating Mytilus species will also be shown. Examples are presented to illustrate the use of 2-DE and MS to differenciate populations, species in taxonomic analysis.