Zaluzianskya (Scrophulariaceae s.s., tribe Manuleeae) is a diverse genus of 55 species of annual and perennial herbs, endemic to southern Africa. This study used DNA sequence data from the nrDNA ITS regions, rpl16, and trnL-trnF to produce the first broad phylogenetic hypotheses for Zaluzianskya, allowing the first tests of the monophyly of the genus and its sections, as well as an examination of character evolution. Reyemia nemesioides was placed within Zaluzianskya with strong support. This is consistent with several morphological characteristics of both species of Reyemia. Sections Zaluzianskya and Holomeria are not monophyletic. The single species sampled from section Macrocalyx (Z. mirabilis) was nested within section Nycterinia according to the chloroplast and combined data, but placed sister to Nycterinia according to the ITS data. Several species do not form clades. Populations of Z. villosa and Z. gracilis were nested within a Z. affinis clade while Z. divaricata was nested within a Z. pusilla clade. Finally, populations of the sole day-flowering species within section Nycterinia, Z. microsiphon, were separated on the phylogenies. The source of this division is unknown but could be hybridization or convergent evolution via pollinator selection. Analyses suggest that the plesiomorphic condition for Zaluzianskya is annual, distributed within arid regions. Subsequent derivations of a perennial life history appear associated with distribution in relatively mesic regions.