The primary structure of the 5.8S rRNA gene was established in 37 species of the genus Nicotiana representing four different sections of the subgenus Petuniodes: Acuminatae, Alatae, Noctiflorae, and Suaveolentes. On the basis of these data, phylogenetic analysis was performed. The obtained results are in good agreement with the modern systematics of the genus. However, the bootstrap analysis gives only poor statistical support for the branches of the phylogenetic tree owing to a small number of phylogenetically informative positions.