Bacteria have been regarded as one of the most important factors in pulpal and periapical diseases. Streptococci are frequently isolated facultative anaerobes in infected root canals. Recently molecular biological techniques have been rapidly progressed. This study was designed to apply the molecular biological tools to the identification and classification of streptococci in the endodontic microbiology. Streptococci isolated from infected root canals were identified with both Vitek Systems and API 20 STREP. Identification results were somewhat different in several strains of streptococci. Eighteen streptococci and enterococcal was difficult so to digest plasmid DNA using Hind III and EcoRI to differentiate strains by restriction enzyme analysis of plasmid DNA. 16S rDNA of chromosome was amplified by polymerase chain reaction(PCR) and then restricition fragment length polymorphism(RFLP) using several restriction enzymes was observed. The molecular mass of 16S rDNA of chromosomal DNA was approximately 1.4kb. There were three to five RFLP patterns using eight restriction enzymes. RFLP patterns digested with CfoI which recognizes four base sequences were identical in all stains. Hind III which recognizes six base sequences could not digest the 16S rDNA. Restriction enzymes which recognize five base sequences were suitable for RFLP pattern analysis. At least three different restriction enzymes were needed to compare each strains. 16S rDNA PCR-RFLP was simple and rapid to differentiate and classify strains and could be used in the epidemiological study of root canal infections.