Objective To investigate the prevalences of common suspected pathogens in root canals from combined periodontalendodontic lesions of periodontal origin. Methods Thirty teeth with periodontal-endodontic lesions of periodontal origin were collected (experiment group), and DNA was extracted from tissues in root canals by paper point after opening endodontic access and rinsing. Five healthy premolars extracted for orthodontics were served as negative controls. Two-step PCR was employed to detect the suspected pathogens, including periodontal suspected pathogens (T. forsythensis, P. gingivalis, F. nucleatum, P. intermedia and T. denticola) and endodontic infection related microorganism (P. endodontalis). Results Target microorganism infection was detected in 13(43.3%) tissue samples in root canals in experiment group. There were 6(20.0%) samples with T. forsythensis infection, 5(17.0%) with P. intermedia infection, 4(13.0%) with P. gingivalis infection, 4(13.0%) with P. endodontalis infection and 1(3.0%) with F. nucleatum infection, while T. denticola was not detected in experiment group. Target microorganisms were not detected in all tissue samples in root canals in control group. Conclusion Periodontal suspected pathogen infection contributes to the majority of combined periodontal-endodontic lesions of periodontal origin.