Recent findings of new genes in fish related to the prion protein (PrP) gene PRNP, including our recent report of SPRN coding for Shadoo (Sho) protein found also in mammals, raise issues of their function and evolution. Here we report additional novel fish genes found in public databases, including a duplicated SPRN gene, SPRNB, in Fugu, Tetraodon, carp, and zebrafish encoding the Sho2 protein, and we use comparative genomic analysis to analyze the evolutionary relationships and to infer evolutionary trajectories of the complete data set. Phylogenetic footprinting performed on aligned human, mouse, and Fugu SPRN genes to define candidate regulatory promoter regions, detected 16 conserved motifs, three of which are known transcription factor-binding sites for a receptor and transcription factors specific to or associated with expression in brain. This result and other homology-based (VISTA global genomic alignment; protein sequence alignment and phylogenetics) and context-dependent (genomic context; relative gene order and orientation) criteria indicate fish and mammalian SPRN genes are orthologous and suggest a strongly conserved basic function in brain. Whereas tetrapod PRNPs share context with the analogous stPrP-2-coding gene in fish, their sequences are diverged, suggesting that the tetrapod and fish genes are likely to have significantly different functions. Phylogenetic analysis predicts the SPRN/SPRNB duplication occurred before divergence of fish from tetrapods, whereas that of stPrP-1 and stPrP-2 occurred in fish. Whereas Sho appears to have a conserved function in vertebrate brain, PrP seems to have an adaptive role fine-tuned in a lineage-specific fashion. An evolutionary model consistent with our findings and literature knowledge is proposed that has an ancestral prevertebrate SPRN-like gene leading to all vertebrate PrP-related and Sho-related genes. This provides a new framework for exploring the evolution of this unusual family of proteins and for searching for members in other fish branches and intermediate vertebrate groups.