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The New World grass genus Bouteloua (Chloridoideae: Cynodonteae) comprises 57 species, 13 of which produce unisexual spikelets and hence are diclinous. Andromonoecy, gynodioecy, monoecy, trimonoecy, and dioecy all occur in the genus, and ten species are known to express more than one of these breeding systems. Employing a phylogenetic estimate based on parsimony analysis of DNA sequences from the ITS (nrDNA) and trnL–F (cpDNA) regions representing 35 species of Bouteloua, including ten of 13 diclinous species, we used parsimony character state reconstructions to investigate the evolution of unisexual spikelets and breeding systems. Our specific goals were to estimate (1) the ancestral condition in the genus, (2) the number of times spikelet unisexuality has arisen and/or been lost, and (3) the evolutionary pathways leading to dioecy and the other breeding systems. Although a number of related genera are diclinous, the reconstructions suggest that the common ancestor of Bouteloua probably was not diclinous. Spikelet unisexuality appears to have evolved two to seven times in the genus, but precisely how many times is uncertain; zero to four reversals to hermaphroditism are inferred. The reconstructions show andromonoecy arising from monoecy, and gynodioecy arising from monocliny. Neither andromonoecy nor gynodioecy are implicated in the origin of monoecy or dioecy. Monoecy is shown to evolve from monocliny and dioecy, and dioecy from monocliny and monoecy. Polyploidy and arid environments are possible factors in the evolution and variability of breeding systems in Bouteloua.