Aims: The goal of this study was to characterize recently identified, non-flowering, putative tetraploid Sorghum spp. hybrids utilizing bulked segregant analysis with SSRs and compare them to S. bicolor, S. halepense, and triploid putative Sorghum spp. hybrids. Confirmed species hybrids between S. bicolor and S. halepense would provide resources for investigating risks of invasiveness and transgene escape alongside potential for identifying novel perennial Sorghum feedstocks of value. Study design: Bulked segregant analysis of Sorghum species and S. species hybrids. Place and Duration of Study: Department of Soil & Crop Sciences; Texas AM College Station, TX, USA; 2009 2011 Methodology: A bulked segregant analysis approach was conducted using SSRs mined from the S. bicolor genome sequence. Bulked samples of S. bicolor, typical flowering S. halepense, non-flowering tetraploid putative Sorghum spp. hybrids, and triploid putative Sorghum spp. hybrids were surveyed to identify both unique markers specific to each bulk and markers indicative of S. bicolor genetic material introgressed into the Sorghum spp. hybrids. Results: Thirty-nine and 23 markers were found to be unique to the S. bicolor and typical flowering S. halepense bulks, respectively. These unique markers could be utilized in breeding programs to identify interspecific hybrids. A subset of 23 unique SSRs were found in the non-flowering tetraploid putative Sorghum spp. hybrid, and they may be useful in the characterization of the non-flowering phenotype. Conclusion: Markers identified in this study provide: 1) species-specific tools for confirmation of interspecific Sorghum spp. hybrids and quantification of gene flow between Sorghum spp., 2) candidate gene and genomic region resources for dissection of the nonResearch Article American Journal of Experimental Agriculture, 2(1): 9-20, 2012 10 flowering phenotype, and 3) Sorghum spp. hybrid-specific markers suitable towards development of perennial biofuel and forage sorghum feedstocks.