Mitotic metaphase chromosomes were counted in 29 taxa, representing 11 subgenera of Austrostipa, and in 11 species from nine related genera of the grass subfamily Pooideae. Karyotype features were also measured. The cytogenetic data were mapped on molecular phylogenetic trees based on nuclear ITS and plastid 3′ trnK DNA sequence data. The trees showed four different main lineages within Austrostipa, but supported only two of the 13 acknowledged subgenera. The phylogenetic positions of the genera Anemanthele, Achnatherum, Nassella and Oloptum indicated paraphyly of the genus Austrostipa. In nuclear-sequence data, Anemanthele was nested within Austrostipa; however, in plastid-sequence data, both were sisters. The newly obtained chromosome counts in Austrostipa showed that most species have 2n = 44, the other 2n = 66. Presuming a chromosome base number of x = 11, the counts corresponded with ploidy levels of 4x and 6x respectively. Karyotype data of Austrostipa and Anemanthele were very similar. Chromosome counting in further genera suggested chromosome base numbers of x = 9, 10, 11, 12 and 13. Chromosome sizes of the phylogenetically derived tribe Stipeae were smaller than those of the earliest diverging Pooideae lineages Nardeae, Meliceae and Phaenospermateae. The mechanisms of chromosome evolution and the origin of the considerable variation in chromosome base numbers in the subfamily Pooideae are discussed in the context of chromosome evolution and biosystematics.