Coral reef ecosystem is known as "tropical rainforest of the ocean" due to its high biodiversity and primary productivity. Bacteria are known to be abundant and active in different coral niches, including coral surface mucus layer, coral tissue, and surrounding reef waters. Coral-associated bacteria play important roles in resisting pathogen, cycling of nitrogen, carbon and sulfur in coral reef ecosystem. Previous studies on coral microbiology in China mainly focused on the genetic diversity of zooxanthellae and their ecological and physiological roles in corals, and so far few investigations have been made in regard to the diversity of coral-associated bacteria, especially coral-associated diazotrophic bacteria. This research aimed to elucidate the nifH gene diversity of nitrogen-fixing bacteria associated with P. damicornis, and compare the nitrogen-fixing bacteria diversity. Three samples (mucus, tissue of coral P. damicornis from Sanya Bay and tissue of coral P.damicornis from Xisha Islands) were investigated, which were named as SYN, SYZ and XSZ, respectively. The nitrogenase gene nifH diversity of three coral samples were assessed using clone library construction in combination with Polymerase Chain Reaction-restriction Fragment Length Polymorphism (PCR-RFLP). Different operational taxonomic unit (OTU) groups (RFLP patterns) were recovered from the different coral samples after digested by the restriction enzymes TaqI and HaeⅢ, respectively. As a result, 23 OTUs were recovered from 155 clones of SYN, 25 OTUs were recovered from 120 clones of SYZ, while 13 OTUs were recovered from 102 clones of XSZ with clone coverage was 90.3%,87.5% and 94.1%, Shannon-Wiener index (H) was 2.28, 3.02 and 2.08, Evenness index (E) was 0.5, 0.65 and 0.56, respectively. The findings of this study showed that 1) Clone libraries demonstrated that the majority of retrieved sequences from coral mucus and tissue libraries were identified to be Chlorobia and Proteobacteria (including α-, γ-and δ-Proteobacteria). 2) Most of OTUs were related to those nifH gene sequences of uncultured bacteria from a variety of habitats in marine environments, such as coral reef, sea sponges, ocean water column and marine sediment, which indicated coral P. damicornis might harbored a large number of new diazotroph resources that had not yet been cultured. 3) The study also found two bacterial group-OTUs related to Chlorobia and Desulfovibrio, which may not be only as pathogenic bacteria as described in the previous reviews, but also played important roles in nutrient cycles, defensing of coral symbionts, they were suspected to resist coral bleaching by fixing nitrogen. 4) Shannon-Wiener index indicated that the diazotrophic diversity of SYN was lower than that of SYZ, and diazotrophic diversity associated with XSZ was lower than that of SYZ, showing that the community diversity of diazotroph assoiated with coral differed based on the geographic location and specific environment characterices of the coral.