Eupatorium ×truncatum (Asteraceae) is distributed throughout the eastern United States and is thought to have originated from interspecific hybridization between E. perfoliatum and E. serotinum. This study confirms the hybrid origin of one E. ×truncatum population in central Indiana through analysis of DNA sequence data from nuclear ITS and corroborates the morphology-based hypothesis that E. perfoliatum and E. serotinum are its progenitors. Moreover, two single-copy nuclear loci showing fixed differences between the parents were in the heterozygous form in all hybrids analyzed, suggesting that these are all F₁ hybrids. Plastid psbA-trnH intergenic spacer sequences identify E. serotinum as the maternal progenitor of all sampled hybrids.