[Background] Many endophytic diazotrophs can be found in gramineous plants and play important roles in promoting plant growth, nutrient utilization and stress resistances. [Objective] This research aims to reveal the composition and variation of endophytic diazotrophs in roots, stems and leaves of Pennisetum sp. at different developmental stages. [Methods] High-throughput sequencing technology was used to analyze the endophytic diazotrophs flora in roots, stems and leaves at different growth stages of Pennisetum sp. [Results] About 40 000 to 60 000 useful sequences were obtained from 15 samples in the roots, stems and leaves of Pennisetum sp. at different growth stages, and were mainly distributed within about 360 bp. At maturity period, the abundance of endophytic diazotrophs from the root of Pennisetum sp. was the highest whereas at jointing period, the stem and leaf samples showed the highest abundance. In the same growth period, the trend in abundances was observed thus: root > leaf > stem. This trend of variation was consistent with the trend of nitrogenase activity in plant samples of Pennisetum sp. The major bacterial communities identified belong to the Proteobacteria, Cyanobacteria and Klebsiella, Herbaspirillum, Bradyrhizobium. As a whole, the composition of the microbial community present in root and leaf samples was similar, whereas that from the stems had a cross with the roots and leaves. At maturity period, the abundance of associated nitrogen-fixing bacteria was highest in the roots. CCA analysis showed that the endophytic diazotrophs from different samples were mainly affected by environmental temperature, followed by moisture and pH. [Conclusion] There were great differences in composition and abundance of endophytic diazotrophs from roots, stems and leaves at different growth stages of Pennisetum sp. Our findings provide important information for development and utilization of endophytic diazotrophs resources from Pennisetum sp.