Genomics has revolutionized our understanding of genetic variability within and among species. The vast amount of information available from genome sequencing is a source for developing new strategies for understanding population dynamics, among other things. However, these resources are generally scarce for non-model species, even those of high economic interest. This chapter describes the most commonly used strategies for sequencing, assembling, and annotating new genomes and how complete or draft genomes have been used to discover new polymorphic sites for studying population genetics in the Neotropics. In this context, this chapter addresses issues related to genome sequencing, including the selection of target species, the best sequencing methods to use considering genome size and financial cost, and the most commonly employed strategies for nuclear genome assembly and annotation. In addition, given the still high cost of sequencing the genomes of new species, this chapter includes examples of how even low-coverage genomic data can be used to develop powerful resources for population genomics studies. Finally, this chapter discusses the multiple uses of newly assembled genomes for unraveling genetic variability within and between species, including their use in molecular marker development. The discussions presented here will provide the reader with the main challenges and strategies to overcome them in studies of neotropical species, especially those still poorly understood.