Abstract: To establish a rapid and accurate method for authenticating hybrid Pennisetum, molecular markers were developed based on specific sequences derived from the genomes of pearl millet（Pennisetum glaucum）and elephant grass（Pennisetum purpureum）. These markers were employed to verify the genetic identity of hybrid Pennisetum, with validation conducted via chromosome counting and genomic in situ hybridization（GISH）on 16 candidate progeny lines. Results demonstrated that comparative sequence analysis of the A subgenome of pearl millet, along with the A' and B subgenomes of elephant grass, yielded 1146, 2421, and 4067 specific sequences, respectively. From these, 21 molecular markers were developed, and three highly efficient markers were ultimately selected. PCR amplification and agarose gel electrophoresis revealed that 13 out of the 16 candidate lines were true allotriploid hybrids, while the lines Minmu No. 6, Reyan No. 4, and Guimu No. 1 were identified as tetraploid elephant grass rather than genuine hybrids. Chromosome counting and GISH results were fully consistent with the molecular marker findings, confirming the reliability of the marker system. This study successfully establishes a robust method for authenticating hybrid Pennisetum and provides a valuable reference for the identification of hybrids in other complex polyploid plant species.