The neotropical region is a highly significant and biodiverse area, spanning through a large portion of Central and South America and encompassing diverse landscapes. The region holds immense global importance for biodiversity, as it offers essential ecological services crucial to the planet’s health. Ensuring the protection of the region and its unique biodiversity is of utmost importance for the well-being of current and future generations. To this end, phylogeography, a relatively recent field, provides a historical and evolutionary context for biodiversity studies since it investigates the historical processes that have influenced genetic diversity. Phylogeography enables the formulation of biogeographic hypotheses, the description of the evolution of reproductively isolated population units, the inference of processes relating to the origin, distribution, and maintenance of biodiversity, and the creation of hypotheses concerning the influence of past environmental changes, physical and biotic characteristics of populations, population structure, and genetic diversity. This chapter presents an overview of how phylogeography can be instrumental in identifying divergent lineages, understanding species’ responses to climatic changes, identifying refuge areas, dating geographical events, and maintaining the neotropical biota. Phylogeography has shed light on high levels of genetic diversity, genetic structure, hybridization, and various historical events, thereby serving as a pillar in population genetics research. Furthermore, advancements in molecular techniques and more efficient machine learning have facilitated the availability of larger datasets for the region, leading to fundamental insights into the evolutionary processes that have given rise to the extensive biodiversity we observe in the Neotropics today.