In this study S. canis and 12 various species and serogroups of streptococci including: S. agalactiae, S. dysgalactiae subsp. dysgalactiae (serogroup C and L), S. dysgalactiae subsp. equisimilis (serogroup G), S. uberis, S. parauberis, S. phocae, S. suis, S. equi subsp. equi, S. equi subsp. zooepidimicus, S. porcinus and S. pyogenes were used and identified reliably by PCR-restriction fragment length polymorphism analysis (PCR-RFLP) of 1.43 kb of 16S ribosomal RNA gene using universal oligonuclotide primers and subsequent digestion with the restriction endonucleases including RsaI, MspI and AvaII. The PCR-RFLP results showed that RsaI restriction RFLP pattern of S. canis appeared different with all streptococci species baring the S. equi subsp. equi and S. equi subsp. zooepidimicus. The MspI restriction RFLP pattern of S. canis could be differentiated from S. agalactiae, S. dysgalactiae subsp. dysgalactiae (serogroups C and L), S. dysgalactiae subsp. equisimilis (serogroups G), S. phocae, S. suis, S. porcinus and S. pyogenes. The AvaII restriction RFLP pattern of S. canis could be distinguished from S. dygalactiae subsp. dysgalactiae (serogroup C and L), S. dygalactiae subsp. equisimilis (serogroup G) S. parauberis, S. phocae and S. suis. In conclusion, PCR-RFLP method using restriction endonucleases RsaI, MspI and AvaII could be useful method for identification of S. canis from S. dysgalactiae subsp. dysgalactiae (serogroup C and L), S. dysgalactiae subsp. equisimilis (serogroup G) and other related streptococci species. It can be concluded that RFLP method might help to determine the prevalence of S. canis in animal and human infections .