Nicotiana species, which share significant similarities with tomato, potato, eggplant, and pepper, originated in South America and subsequently spread to North America, Africa, and Australia. They are known to produce and accumulate alkaloids as part of their defense mechanism against insects and herbivores. Since their description and classification by Goodspeed in 1947, research on Nicotiana species has been ongoing, including investigations on how the genus evolved and its relationship with other Solanaceae members. The presence of diploid and polyploid species in Nicotiana and their large genome sizes resulted in the generation of multiple resources aiming at better understanding of how polyploids formed and evolved over time. The use of Nicotiana tabacum and Nicotiana benthamiana as model organisms in plant research further helped increase the interest in understanding the molecular mechanisms in these species. Here, we review the literature and summarize what is currently known about the phylogeny, cytology, and genetics of Nicotiana species. We also present the current state of the Nicotiana genomes and introduce the functional genomics insights they have provided.