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Pugionium (Brassicaceae) is a small genus that occurs in central Asian deserts. The interspecific delimitation and taxonomic treatments of this genus are disputed and its phylogenetic origin remains unknown. In the present study, we examined these issues based on morphological and molecular data obtained for the first time. We used statistical methods to examine inter- and intraspecific morphological variations. The results suggest that only two species, namely P. dolabratum and P. cornutum, can be warranted for all examined populations and specimens, whereas three species (P. calcaratum, P. cristatum, and P. pterocarpum) should be incorporated into P. dolabratum. This delimitation was further supported by the molecular data: all populations of P dolabratum, P calcaratum, P cristatum, and P pterocarpum shared the same internal transcribed spacer genotype, whereas those from P cornutum had another type. Phylogenetic analyses of Pugionium and representative genera of Brassicaceae based on ndhF sequences suggest that this genus is sister to the genus Megacarpaea, which, together, comprise a well-supported lineage with Farsetia, Lobularia, Iberis, and Ionopsidium, whereas the two other genera that were previously suggested to be closely related to this genus (Isatis and Bunias) were placed in the other lineages. We further discuss the origin of Pugionium and suggest that it probably originated in central Asia when the climate became drier from the late Miocene.