Abstract: Intrageneric relationships in the genus Nicotiana were investigated by comparison of DNA sequences of the matK gene of the chloroplast genome. A total of 40 taxa were examined in this study, representing 39 of the approximately 70 wild species of this genus. We obtained the full sequences of the 1530 bp matK ORFs; no variations in length due to insertions or deletions were found. The phylogenetic trees obtained from maximum parsimony (MP) and neighbour-joining (NJ) methods were fundamentally consistent. The genus Nicotiana formed a clade in these trees. The traditional classification of this genus was mostly in agreement with the molecular phylogeny. However, all three subgenera and some sections did not form a monophyletic group. Character-state mappings were used to infer a centre of origin, biogeographic history, and transition of chromosome number. The results support the previous hypothesis that this genus originated in South America and subsequently spread to other continents. The suggestion that the ancestral basic chromosome number is x = 12 and that polyploidy and aneuploidy have occurred independently several times during the evolution of Nicotiana species is also discussed.