The invariant chain (Ii) is the critical third chain required for the MHC class II heterodimer to be properly guided through the cell, loaded with peptide, and expressed on the surface of antigen presenting cells. Here, we report the isolation of the nurse shark Ii gene, and the comparative analysis of Ii splice variants, expression, genomic organization, predicted structure, and function throughout vertebrate evolution. Alternative splicing to yield Ii with and without the putative protease-protective, thyroglobulin-like domain is as ancient as the MHC-based adaptive immune system, as our analyses in shark and lizard further show conservation of this mechanism in all vertebrate classes except bony fish. Remarkable coordinate expression of Ii and class II was found in shark tissues. Conserved Ii residues and cathepsin L orthologs suggest their long co-evolution in the antigen presentation pathway, and genomic analyses suggest 450 million years of conserved Ii exon/intron structure. Other than an extended linker preceding the thyroglobulin-like domain in cartilaginous fish, the Ii gene and protein are predicted to have largely similar physiology from shark to man. Duplicated Ii genes found only in teleosts appear to have become sub-functionalized, as one form is predicted to play the same role as that mediated by Ii mRNA alternative splicing in all other vertebrate classes. No Ii homologs or potential ancestors of any of the functional Ii domains were found in the jawless fish or lower chordates.