Of the 340 genera in the Brassicaceae, apomictic reproduction is found only in the North American genus Boechera. We investigated phylogenetic relationships, ability to hybridize, mating system, and ploidy levels of 92 lines sampled from 85 populations and representing 19 Boechera species. Phylogenetic analyses based on chloroplast DNA sequences identified three lineages in the genus. Reciprocal crosses of each line were made to a common sexual diploid B. stricta tester. The resulting F(1) progeny were analyzed for the inheritance of polymorphic microsatellite loci, genome size, and seed production. Intraspecific B. stricta crosses confirmed that this species is mostly diploid and sexual. Interspecific crosses revealed many other species were diploid and sexual and could be successfully hybridized with the tester. We also found obligate and facultative apomictic diploid and triploid lines. De novo F(1) polyploids (either triploids or tetraploids) were derived from the union of nonreduced (from an apomictic parent) and reduced (from the tester) gametes. However, seed production of these F(1) plants was generally low, suggesting a failure in the transmission of apomixis. The creation of a wide array of segregating genetic populations will facilitate future research on the evolution and inheritance of quantitative variation in Boechera.