Abstract:[Objective] To explore the diversity and differentiation of endophytic nitrogen-fixing bacteria community in Pennisetum sp. in different habitats. [Methods] High-throughput sequencing of nitrogen-fixing enzyme nifH target gene method was used to study the endophytic nitrogen-fixing bacteria in Pennisetum sp. in 6 typical areas of China, including Fujian Minhou County, Xinjiang Moyu County, Inner Mongolia Alashan Zuoqi, Qinghai Guide County, Gansu Anding District, and Hainan Nada Town. The environmental driving mechanism of nitrogen-fixing bacteria diversity was analyzed by combining geographical and climatic factor statistics. [Results] A total of 64122 valid sequences of nifH genes were obtained, 640 OTUs belonging to 6 phyla, 10 classes, 17 orders, 24 families, 33 genera and 39 species. The species and abundance of predominant endophytic nitrogen-fixing bacteria in Pennisetum sp. from different regions were quite different. At the phylum level, Proteobacteria were the dominant bacteria in the five areas of Fuzhou Minhou County, Gansu Anding District, Xinjiang Moyu County, Inner Mongolia Alashan Zuoqi and Qinghai Guide County, whereas Proteobacteria and Cyanobacteria were the dominant phylum in Hainan Nada town. At the genus level, the most dominant endogphytic nitrogen-fixing bacteria groups in Pennisetum sp. in different regions as below:Minhou County of Fuzhou province (undetermined genus of Proteobacteria, 80.56%), Moyu County of Xinjiang province (undetermined genus of Proteobacteria, 33.14%), Alashan Zuoqi of Inner Mongolia (undetermined genus of Proteobacteria, 76.23%), Anding District of Gansu Province (undetermined genus of Alphaproteobacteria, 53.78%), Nada Town of Hainan (undetermined genus of Proteobacteria, 38.79%), Guide County of Qinghai (undetermined genus of Proteobacteria, 46.12%). The analysis of alpha-diversity and beta-diversity showed that the diversity of endophytic nitrogen-fixing bacteria communities in Pennisetum sp. in different regions was different. The diversity and richness of endophytic nitrogen-fixing bacteria in Pennisetum sp. in Nada Town of Hainan Province were the highest, while those in Minhou County of Fujian province were the lowest. The canonical correspondence analysis (CCA) showed that annual average rainfall and annual average temperature were the main factors affecting the change of endophytic nitrogen-fixing bacteria in Pennisetum sp., followed by soil organic matter, soil total nitrogen and soil pH. [Conclusion] The composition and abundance of endophytic nitrogen-fixing bacteria community in Pennisetum sp. in different areas were quite different. The species and relative abundance of endophytic nitrogen-fixing bacteria in Pennisetum sp. in Nada town of Hainan province were relatively high. This research can provide theoretical supports for the development of endophytic nitrogen-fixing bacteria in Pennisetum sp. and the breeding and production of nitrogen-fixing microbial fertilizers.