The purpose of this study was to investigate and compare the frequence of 4 periodontal pathogens in the supra- and subgingival plaque in periodontally healthy subjects. Twenty adult individuals aged 22 to 28 years (mean age 23.65 years) participated in this study. All subjects had no pocket sites more than 3 mm deep, and the sites selected for sampling were all negative for bleeding. After drying and isolation of the sites with cotton rolls, supragingival plaque was sampled using sterile periodontal curette. Each plaque sample was placed in individual tubes containing 500 ml of 1X PBS. After removal of the supragingival sample and any remaining supragingival plaque, subgingival plaque samples were taken from the same sites using sterile curette and placed in similar individual tubes. Identification of 4 putative periodontal pathogens from the samples was performed by polymerase chain reaction based on 16S rDNA. Chi-square test was employed to identify significant explanatory variables for the presence of the 4 periodontal pathogens. The data show that Actinobacillus actinmycetemcomitans, Porphyromonas gingivalis, Bacteroides forsythus, and Fusobacterium nucleatum occurred in 16.9%, 14.4%, 52.5%, and 80.6%, respectively. No significant differences were noted in the periodontal pathogens between supra- and subgingival plaques according to the kind of teeth. However, the incisors were at higher risk for harboring F. nucleatum (p < 0.05). Conclusion: These results reveal that anaerobic periodontal pathogens can be detected in supragingival plaques. Supragingival plaque may function as a reservoir of periodotopathogens.