Evolutionary biologists and geneticists have long recognized that genetic variation is a keystone for evolution to follow. That said, we cannot properly understand evolution if we do not know the levels and organization of the genetic variation that populations and species have. Population genetics is the field of genetics that deals with the genetic variation and their underlying factors within and among populations. Researchers in the field have amassed data on allele diversity and variation for countless species of animals, plants, fungi and other organisms to which has helped elucidate how much variation they have across their genomes. A brief history of the field discusses how population genetics theory was formulated based on genetic variation inferred from morphological traits and rapidly moved forward to addressing genetic variation at the molecular level. Technological inputs led a cascade of consecutive methods to detect and quantify allele frequencies, beginning with enzymes and their isoforms and culminating with studying variation at the DNA molecule itself. The so-called molecular markers have revealed singularities for the most diverse species across all continents, including organisms of the neotropical biogeographic realm, that includes South America and part of Central America. The increasing use of genome-based approaches should improve our knowledge on population genetic variation of all kinds of organisms, and how such variation is truly important for species adaptation in a changing world, particularly in the hyperdiverse Neotropics.