The vertebrate proglucagon gene encodes glucagon, and the two glucagon-like peptides GLP-1 and GLP-2. To better understand the origin and diversification of the distinct hormonal roles of the three glucagon-like sequences encoded by the proglucagon gene, we have examined the evolution of this gene. The structure of proglucagon has been largely maintained within vertebrates. Duplication of the proglucagon gene or duplications of sequences within the proglucagon gene are rare. All proglucagon gene duplications are likely to be the result of genome duplication events. Examination of the rates of amino acid sequence evolution of each hormone reveals that they have not evolved in a uniform manner. Each hormone has evolved in an episodic fashion, suggesting that the selective constraints acting upon the sequence vary between, and within, vertebrate classes. Changes in selection on a sequence often reflect changes in the function of the sequence, such as the change in function of GLP-1 from a glucagon-like hormone in fish to an incretin in mammals. We found that the GLP-2 sequence underwent rapid sequence evolution in the early mammal lineage, therefore we have concluded that mammalian GLP-2 has acquired a new biological function that is not found in other vertebrates. Comparisons of the hormone sequences show that many amino acid residues that are functionally important in mammalian hormones are not conserved through vertebrate evolution. This observation suggests that the sequences involved in hormone action change through evolution.