Lake trout are used as bioindicators for toxics exposure in the Great Lakes ecosystem. However, there is no knowledge about lake trout proteome. Here we performed the first lake trout (Salvelinus namaycush) liver proteomics and searched the databases against (NCBI and UniProtKB) Salvelinus, Salmonidae, Actinopterygii and the more distant Danio rerio. In the NCBI search, we identified 4371 proteins in 1252 clusters. From these proteins, we found 2175 proteins in Actinopterygii 1253 in Salmonidae, 69 in Salvelinus and 901 in Danio rerio NCBI searches. In the UniProtKB search, we identified 2630 proteins in 1100 clusters. From these proteins, we found 317 in Actinopterygii, 1653 in Salmonidae, 37 in Salvelinus and 666 in Danio rerio UniProtKB searches. A similar outcome was also obtained from a technical replicate experiment. A large number of lake trout liver proteins were not in any Salvelinus databases, suggesting that lake trout liver proteins have homologues to some proteins from the Salmonidae family and Actinopterygii class, as well as to the species Danio rerio, a more highly studied Cypriniformes fish. Therefore, our study not only builds the first comprehensive lake trout protein database, but also establishes protein homology-based evolutionary relationships between the fish within their family and class, as well as distant-related fish (lake trout and zebrafish). In addition, this study opens the possibility of identifying evolutionary relationships (i.e. adaptive mutations) between various groups (i.e. zebrafish, Salmonidae, Salvelinus and lake trout) through evolutionary proteomics