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Objective: The aim of this study was to investigate subgingival infection frequencies ofPorphyromonas gtngivalis and Actinobacillus actinomycetemcomitans strains with genetic variation in Chinese chronic periodontitis (CP) patients and to evaluate its correlation with clinical parameters. Methods: Two multiplex polymerase chain reaction (PCR) assays were developed to detect the 16SrDNA, collagenase (prtC) and fimbria (fimA) genes of P. gingivalis and the 16SrDNA, leukotoxin (lktA) and fimbria-associated protein (lap) genes ofA. actinomycetemcomitans in 60 sulcus samples from 30 periodontal healthy subjects and in 122 subgingival plaque samples from 61 patients with CP. The PCR products were further T-A cloned and sent for nucleotide sequence analysis. Results: The 16SrDNA, prtC andfimA genes ofP. gingivalis were detected in 92.6%, 85.2% and 80.3% of the subgingival plaque samples respectively, while the 16SrDNA, lktA andfap genes ofA. actinomycetemcomitans were in 84.4%,75.4% and 50.0% respectively. Nucleotide sequence analysis showed 98.62%～100% homology of the PCR products in these genes with the reported sequences. P. gingivalis strains with prtC+/fimA+ and A. actinomycetemcomitans with lktA+ were predominant in deep pockets (＞6 mm) or in sites with attachment loss ≥5 mm than in shallow pockets (3～4 mm) or in sites with attachment loss ≤2 mm (P＜0.05). P. gingivalis strains with prtC+/fimA+ also showed higher frequency in gingival index (GI)=3than in GI= 1 group (P＜0.05). Conclusion: Infection of P. gingivalis with prtC+/fimA+ and A. actinomycetemcomitans with lktA+correlates with periodontal destruction of CP in Chinese. Nonetheless P. gingivalis fimA, prtC genes and A. actinomycetemcomitans IktA gene are closely associated with periodontal destruction, while A. actinomycetemcomitansfap gene is not.