ABSTRACTThe mammalian gene Brain-Derived Neurotrophic Factor (BDNF) is an essential mediator of brain assembly, development, and maturation which has been implicated in a variety of brain disorders such as neurodevelopmental disorders (e.g. autism spectrum disorder), neuropsychiatric disorders (e.g. depression, PTSD, schizophrenia), and neurodegenerative disorders (e.g. Parkinson’s). Loss of BDNF during early development is embryonic lethal, and depletion of BDNF during adolescence or adulthood can result in disease-related neuropathology across a broad range of model organisms. In order to better understand the role of BDNF in disease, we seek to provide an evolutionary context to BDNF’s role within the brain by elucidating the molecular and genetic comparative history of BDNF across species. We conduct sequence alignment and phylogenetic reconstruction of the BDNF gene across a diverse selection of over 160 mammalian species spanning ∼177 million years of evolution. Selective evolutionary change was examined via several independent computational models of codon evolution including FEL, MEME, and BGM. We report strict purifying selection in the main functional domain of BDNF (NGF domain, essentially comprising the mature BDNF protein). Specifically, we discover 6 sites in our homologous alignment which are under episodic selection in the early regulatory region of BDNF (i.e. the prodomain) and 23 pairs of coevolving sites that are a part of complex spatial relationships that are distributed across the entire BDNF gene. Thus, we propose that our discovery of both local and distal sites of co-evolution within the pro- and mature-domains of BDNF that likely reflect the evolutionary fine-tuning of BDNF’s unique and complex regulatory capacities whilst also retaining it’s core yet diverse ontogenic functionality within the central nervous system. This discovery consequently supports the idea that the BDNF prodomain is more prone to change than the mature domain, however the fact that this region has also been subject to negative purifying selection also highlights genetic sensitivity and thus partially explains the prodomain’s disease relevance (e.g. Val66Met and other variants) to numerous neuropsychiatric disorders.HIGHLIGHTSWe extracted coding sequences for Brain-Derived Neurotrophic Factor (BDNF) from over 160 mammalian genomes that span approximately ∼177 million years of evolution.We observe strict purifying selection in the main functional domain (NGF) of the BDNF gene in mammals.We observe novel results with 6 sites in our homologous alignment which are under episodic selection in the early regulatory region of BDNF (i.e. the prodomain).We observe 23 pairs of coevolving sites within BDNF. Many of which are a part of complex spatial relationships and are distributed across the entire BDNF gene.These data define exactly how “BDNF is highly conserved” by defining exactly where and how the mammalian BDNF has evolved, confirming the widespread belief that the BDNF prodomain is more prone to change than the mature BDNF protein.