A mass based approach has been advanced to enable mutations associated with the evolution of proteins to be both charted and interrogated using phylogenetic trees built solely from the masses of peptides generated upon protein proteolysis. The modified MassTree algorithm identifies and displays all such mutations and calculates the frequency of a particular mutation across a tree. Its significance in terms of its position(s) on the tree is scored, where mutations that occur toward the basis of the tree are weighted more favourably. A comparison with data generated from a conventional sequence based tree demonstrates the reliability of mutational analysis employing this approach. Although illustrated for the study of the evolution of influenza hemagglutinin in this work, the approach has far broader applicability and can be applied to investigate the evolution of any organism. In the case of simple microorganisms this can be achieved even without the separation of component proteins. Given the central role that mass map or fingerprint data plays in protein identification in proteomics, this work demonstrates that such data can be successfully employed in a phylogenetics strategy to better understand and predict future evolutionary trends from the perspective of functional proteins expressed by the organism.