BackgroundThe introduction of DNA-based molecular markers made a revolution in biological systematics. However, in cases of very recent divergence events, the neutral divergence may be too slow, and the analysis of adaptive part of the genome is more informative to reconstruct the recent evolutionary history of young species. The advantage of proteomics is its ability to reflect the biochemical machinery of life. It may help both to identify rapidly evolving genes and to interpret their functions.MethodsHere we applied a comparative gel-based proteomic analysis to several species from the gastropod family Littorinidae. Proteomes were clustered to assess differences related to species, geographic location, sex and body part, using data on presence/absence of proteins in samples and data on protein occurrence frequency in samples of different species. Cluster support was assessed using multiscale bootstrap resampling and the stability of clustering—using cluster-wise index of cluster stability. Taxon-specific protein markers were derived using IndVal method. Proteomic trees were compared to consensus phylogenetic tree (based on neutral genetic markers) using estimates of the Robinson–Foulds distance, the Fowlkes–Mallows index and cophenetic correlation.ResultsOverall, the DNA-based phylogenetic tree and the proteomic similarity tree had consistent topologies. Further, we observed some interesting deviations of the proteomic littorinid tree from the neutral expectations. (1) There were signs of molecular parallelism in twoLittorariaspecies that phylogenetically are quite distant, but live in similar habitats. (2) Proteome divergence was unexpectedly high between very closely relatedLittorina fabalisandL. obtusata, possibly reflecting their ecology-driven divergence. (3) Conservative house-keeping proteins were usually identified as markers for cryptic species groups (“saxatilis” and “obtusata” groups in theLittorinagenus) and for genera (LittorariaandEchinolittorinaspecies pairs), while metabolic enzymes and stress-related proteins (both potentially adaptively important) were often identified as markers supporting species branches. (4) In all fiveLittorinaspecies British populations were separated from the European mainland populations, possibly reflecting their recent phylogeographic history. Altogether our study shows that proteomic data, when interpreted in the context of DNA-based phylogeny, can bring additional information on the evolutionary history of species.