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Guayule (Parthenium argentatum A. Gray) is a perennial woody shrub native to the North American Chi-huahuan Desert that holds promise as a sustainable source of natural rubber and hypoallergenic latex. The improvement of guayule for commercial-scale production could be accelerated through genomics-assisted breeding, but such a strategy is severely limited by the paucity of available genomic tools and well-characterized genetic resources. To that end, we used genotyping-by-sequencing (GBS) to simultaneously identify and genotype tens of thousands of single-nucleotide polymorphism (SNP) markers across 62 plant samples from seven wild and cultivated guayule, three Parthenium interspecific hybrid, four mariola (Parthenium incanum Kunth), and one wild quinine (Parthenium integrifolium L) accession(s) that have been characterized for ploidy level and nuclear genome size in this and a prior study. Phylogenetic analysis using the SNP data identified at least two distinct sources of guayule breeding material in the cultivated accessions, previously unknown multi-species hybrids within two Parthenium hybrid cultivars, and guayule/mariola hybrids within one guayule and one mariola wild collected accession. Similar to previously reported results for guayule and mariola, we observed aneuploidy and multiple ploidy levels among individual plants (mixed ploidy) within three Parthenium interspecific hybrid accessions newly characterized in this study. Nuclear genome size characterization of wild quinine, a first for this species, found an estimated haploid nuclear genome size (5757 Mb) for thetetraploid (2n =4x = 72) accession that was more than 1.5-fold larger than that of tetraploid (2n = 4x= 72) guayule or mariola. Together, these results further underscore the need for a comprehensive characterization of available guayule germplasm and sister taxa with both SNP markers and flow cytometry, illustrate the novel utility of GBS for the genus Parthenium, and lay the foundation for genomics-assisted breeding in guayule. (C) 2015 Elsevier B.V. All rights reserved.