Background <p>Retrotransposons play outsized roles in the evolution of gene regulation, genome function, and disease pathogenesis, and more recently they have sparked interest as instruments for new gene therapy approaches. R2 retrotransposons insert site-specifically into the multicopy genes encoding 28S ribosomal RNA at a target sequence conserved broadly across eukaryotic evolution. R2 retrotransposons have been detected in many animals, but previous surveys have been limited in scope and methodology.</p> Results <p>Here, we substantially expand the known distribution of R2 retrotransposons from previously unrepresented or underrepresented taxonomic groups, ranging from ctenophores to amphibians and reptiles. We discover diverse R2 domain architectures and motifs and identify many new avian R2 candidates for genome engineering development. Overall, phylogenetic analyses reveal two highly successful R2 lineages. We describe lineage-distinctive features of the N-terminal DNA recognition domains and reverse-transcriptase domain signatures. Within a lineage, R2 protein sequences do not necessarily preserve the unifying configuration of N-terminal domains assumed in the current clade classification scheme. We show that recombinant R2 proteins with distinctive domain architectures and distribution across major animal classes support target-primed reverse transcription with conserved site specificity.</p> Conclusions <p>Our analysis of the surprisingly varied domain architectures that support target-site specificity informs new R2 classification criteria and provides a greatly expanded foundation for additional structure/function insights about DNA binding selectivity. This expanded perspective on R2 evolution informs approaches for engineering therapeutic gene insertion technologies and offers an opportunity to investigate the conservation and diversification of retrotransposons.</p>