Root nodules of Hippophae rhamnoides were collected in the Maxian Mountains. The plate culture method and high-throughput sequencing were used to study the diversity of endophytic bacteria in the root nodules of Hippophae rhamnoides. Microbial composition, abundance, and diversity of the nodule endophytic bacteria were analyzed. The differences in the results of the two techniques were compared. The results showed that 24 phyla, 50 classes, 90 orders, 167 families, and 215 genera of nodule endophytic bacteria were detected by high-throughput sequencing, and 3 phyla, 5 classes, 7 orders, 8 families, and 8 genera were obtained by plate culture. The relative abundances of the dominant nodule endophytic bacteria revealed that the differences between the results of the two techniques were greater at lower classification levels, especially at the family and genus levels, than at higher levels. Both plate culture and high-throughput sequencing showed an abundant diversity of nodule endophytic bacteria. High-throughput sequencing provides a more comprehensive and accurate picture of microbial community structure than the plate culture method does. Plate culture can only detect a few dominant taxa of nodule endophytic bacteria. The results of this study indicated that plate culture greatly underestimates microbial community composition and overestimate their abundance.