During the last decades, the use of biochemical and PCR-based molecular markers has revealed genetic variation at both the protein and DNA levels for several neotropical organisms, playing an important role in population genetic studies in the Neotropics. Taking into account the crucial importance of standing genetic variation (i.e., genetic diversity) in conservation and management policies, here we describe the use of biochemical (i.e., allozyme) and PCR-based molecular markers (e.g., amplified fragment length polymorphism—AFLP, simple sequence repeats—SSR, inter simple sequence repeats—ISSR, and single nucleotide polymorphism—SNP) to assess intra- and inter-population genetic diversity in a plethora of tropical species. Although numerous advances on population genetics methods and computational tools for their analyses have been made, the development of modern molecular methods (eg, high-throughput DNA sequencing methods to identify SNPs) could be sometimes misunderstood, resulting in an inappropriate choice of the molecular marker that is sometimes more expensive and labor intense than others. Here, we emphasize that a straightforward experimental design encompassing eminent questions/hypotheses must be aligned with the adequate choice of the molecular marker technology, that in turn is sample size-dependent. We also proposed to discuss the use of bi- and polyallelic-based markers, dominantly or co-dominantly inherited, to investigate the genetic diversity patterns in tropical species with distinct life history traits and ecological requirements. We summarize the genetic diversity patterns in a few plants and forest trees as examples, following the timeline of the molecular markers evolution, and depicting fundamental population genetics questions related to conservation genetics from a fine spatial scale perspective to a landscape genetics scenario.