Nierembergia (Solanaceae) contains 21 species found primarily in South America, with one species occurring disjunctly in Mexico. Among other features, a pollination system not found elsewhere in the Solanaceae typifies this genus. In most species, nectaries are absent, but oil-producing elaiophores are present on the corolla limb and these attract oil-collecting bees. Molecular phylogenetic hypotheses based on nuclear ITS and chloroplast rpl16 intron data support the monophyly of Nierembergia, which is sister to a clade composed of Bouchetia plus Hunzikeria. The data reconstruct two clades within the genus: one composed primarily of herbaceous species with broad stigmas and the second containing woody species with crescent-shaped stigmas. Morphological homoplasy is frequent among the species of the herbaceous clade, while the woody clade is morphologically cohesive. Chromosome counts are confirmed and karyotype features are given for 13 Nierembergia species (including six varieties) and for Bouchetia anomala, Leptoglossis linifolia, and Petunia axillaris. The two clades are also supported by karyotypic features: one has asymmetrical karyotypes, small chromosomes, and two chromosome pairs with nucleolar organizing regions (NOR) and the other has symmetrical karyotypes, large chromosomes, and only one NOR.