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Neuroscience is enjoying a renaissance of discovery due in large part to the implementation of next-generation molecular technologies. The advent of genetically encoded tools has complemented existing methods and provided researchers the opportunity to examine the nervous system with unprecedented precision and to reveal facets of neural function at multiple scales. The weight of these discoveries, however, has been technique-driven from a small number of species amenable to the most advanced gene-editing technologies. To deepen interpretation and build on these breakthroughs, an understanding of nervous system evolution and diversity are critical. Evolutionary change integrates advantageous variants of features into lineages, but is also constrained by pre-existing organization and function. Ultimately, each species’ neural architecture comprises both properties that are species-specific and those that are retained and shared. Understanding the evolutionary history of a nervous system provides interpretive power when examining relationships between brain structure and function. The exceptional diversity of nervous systems and their unique or unusual features can also be leveraged to advance research by providing opportunities to ask new questions and interpret findings that are not accessible in individual species. As new genetic and molecular technologies are added to the experimental toolkits utilized in diverse taxa, the field is at a key juncture to revisit the significance of evolutionary and comparative approaches for next-generation neuroscience as a foundational framework for understanding fundamental principles of neural function.