Sixty-nine bovine mastitis streptococci belonging to the species Str. agalactiae (n = 13), Str. dysgalactiae (n = 16), Str. canis (n = 22), Str. uberis (n = 20) and Str. parauberis (n = 4) and six reference strains of the five streptococcal species were examined for their tRNA gene intergenic length polymorphism (tDNA-ILP) fingerprint pattern. Epidemiologically unrelated isolates from bovine mastitis cases were selected by macrorestriction analysis with pulsed-field gel electrophoresis (PFGE). Their results were compared with those obtained from biochemical and serological studies and with those obtained by PCR-mediated identification amplifying species-specific gene segments of the five streptococcal species. According to the present results tDNA-ILP allowed a correct identification of all Str. agalactiae, Str. uberis and Str. parauberis strains investigated also including the reference strains of each species showing species-specific banding pattern. However, all Str. dysgalactiae ssp. dysgalactiae and all Str. canis strains appeared with an undistinguishable pattern which did not allow an identification of the species.