ObjectiveTo characterize the subgingival microbiota in subjects with stage I/II periodontitis (moderate periodontitis, MP), stage III/IV periodontitis (severe periodontitis, SP), and periodontal health (PH) at the same probing depth (PD) (shallow <= 3 mm, moderate 4-6 mm, or deep >= 7 mm), and to investigate the changes associated with probing depth progression. Materials and methods100 subgingival plaque samples were collected from 50 subjects (16 MP, 17 SP and 17 PH), forming six groups: PHS (PH, shallow), MPS (MP, shallow), MPM (MP, moderate), SPS (SP, shallow), SPM (SP, moderate), and SPD (SP, deep). Samples were analyzed using high-throughput sequencing. ResultThe subgingival microbiome showed significant differences associated with both PD and periodontitis stage (p < 0.05). With increasing PD, alpha diversity initially increased and then decreased. Pathogenic genera like Fusobacterium, Filifactor, and Porphyromonas increased, while health-associated genera like Streptococcus and Haemophilus decreased. At shallow sites, the PHS, MPS, and SPS groups showed similar community structure. At moderate and deep sites, the SPM and SPD groups exhibited significant differences in community structure compared to the MPM group, with the SPM and SPD groups showing decreased abundances of Actinomyces and increased abundances of Treponema. The microbial co-networks in the SPD and SPM groups exhibited greater complexity and connectivity and were more resilient to random microbial or node removal. ConclusionsThe subgingival microbiome shows strong associations with PD and periodontitis stage. Clinical relevanceOnce periodontitis progresses to stage III/IV, reconstructing a healthy subgingival microbiome may be challenging, emphasizing the importance of early prevention.