Root nodules are the symbiotic structure formed by microorganisms invading the roots of host plants. These microorganisms can be collectively referred to endophytes. The endophytic bacteria in the nodules of leguminous plants are often called rhizobia, while the actinomycetes are the main bacteria that infect non-leguminous plants to form nodules. The main endophytic actinomycetes in non-leguminous plants is Frankia spp.. These non-leguminous plants are also known as actinorhizal plants. Hippophae tibetana is a typical actinorhizal plant. It is very important to study the endophytes of H. tibetana nodules because of the particularity of H. tibetana distribution habitat. For the study of endophytes from H. tibetana root nodules, the culture method is difficult to simulate the natural conditions and it is difficult to obtain pure culture, and the high-throughput sequencing technology provides convenience for the study of their diversity. In this study, the root nodules of H. tibetana, which grew in Jinqiang River beach in Tianzhu County, Gansu Province, were used as the material. The 16S rRNA gene amplification high-throughput sequencing method was adopted to analyze the diversity of Frankia and non-Frankia endophytic bacteria associated with H. tibetana root nodules combined with Operational Taxonomic Units (OTUs). The results showed that the endophytes of H. tibetana root nodules had a rich diversity. Frankia was single dominant genus comprising of 47.63% of the relative abundance of total endophytes, and a total of 7 OTUs of Frankia were detected in H. tibetana root nodules. In addition to Frankia, there were also a large number of non-Frankia endophytic bacteria in H. tibetana root nodules. A total of 1523 OTUs of non-Frankia endophytes were detected, belonging to 22 phyla, 33 classes, 69 orders, 113 families, and 202 genera. There are 25 non-Frankia OTUs among the top 9 genera in relative abundance. This research also revealed that there was rich diversity of endophytes associated with root nodules of H. tibetana. There was not only Frankia but also non-Frankia in H. tibetana root nodules. There were different species of Frankia in the same nodule sample. This study can provide reference for the analysis of Frankia spp. diversity based on high-throughput sequencing and enrich the database of host plants diversity and Frankia species diversity. This study not only expanded the methods of studying the diversity of endophytic bacteria in H. tibetana root nodules, but also provided an analytical approach for the diversity of Frankia spp. in the same host plant.