First coined in the 1980s to refer to the biological diversity existing on Earth, the term “biodiversity” is currently defined at three different levels: genetics, species and ecosystems. Biological diversity exists because genetic changes made it possible for organisms to adapt in the most diverse environments. The biodiversity on Earth is distributed across biogeographic regions, which include the Neotropics, comprising both Central and South America. The Neotropics have the highest biodiversity richness in the world, and are characterized by their tropical rainforests, including the Amazon Forest, which is home to the greatest species diversity on Earth. Despite this huge biodiversity, the Neotropics as well as other regions on the planet have been dramatically affected by human activities, and biodiversity loss is one of the most pressing concerns in the Anthropocene. Populations adapted to very specific conditions are being locally extinct, depleting species diversity and increasing the risk of species extinction. By assessing the frequency of alleles in populations and their changes associated with mutation, genetic drift, natural selection, and gene flow, population genetics is a powerful tool to evaluate the effects of the Anthropocene on the long-term persistence of species. Nevertheless, neotropical species, and their populations and diversification process are still poorly studied. This chapter highlights key questions about the importance of studying and conserving neotropical biodiversity, indicating that population genetics can provide essential information for biological conservation, contributing to the sustainability and maintenance of life on Earth, one of the greatest challenges of contemporary human society.