OBJECTIVE To investigate the distribution of H. actinomycetemcomitans, P. gingivalis, P. intermedia, T. forsythensis and T. denticola in the subgingival plaque in the patients with chronic periodontitis. METHODS 27 patients with chronic periodontitis were included. Two of the deepest pockets of each patient were selected as the study sites. Semi-quantification of subgingival microorganism samples was analyzed by multiplex polymerase chain reaction and reverse hybridization assay. RESULTS P. gingivalis, P. intermedia, T. forsythensis and T. denticola were detected in a high proportion of examined sites(corresponding values were 98.15%, 92.59%, 100% and 98.15%), however, the proportion of H. actinomycetemcomitans was low (20.37%). The levels of P. gingivalis and T. forsythensis were higher than the other three microorganisms with statistical significance. CONCLUSION The simultaneous infection of P. gingivalis, T. forsythensis, P. intermedia and T. denticola is found in the patients with chronic periodontitis, in which the levels of P. gingivalis and T. forsythensis are higher than the other two microorganisms.