Interspecific hybridization of Palmer amaranth and common waterhemp produce hybrids with unique DNA fragments not found in either parent. The objective of this research was to investigate the mechanisms involved in the formation of the polymorphic fragments. Six novel fragments were cloned and sequenced. Five of the six were significantly similar to plant transposons, the sixth was similar to squamosa promoter–binding proteins from other plant species. Southern blot analysis using one of the novel fragments as probe revealed a consistent pattern of repetitive DNA that was species and biotype specific. These results indicate that transposon-like elements may play an important role in the formation of new fragments in Amaranthus hybrids derived from interspecific hybridization, suggesting that considerable instability of the hybrid genome may occur.