Pulsed-field gel electrophoresis (PFGE) was examined as a tool to differentiate strains of streptococci isolated from clinical and sub-clinical cases of bovine mastitis. Analysis of SmaI chromosomal digests of Streptococcus agalactiae, S. dysgalactiae subsp. dysgalactiae and S. uberis isolates revealed intraherd and interherd strain relationships within each species. Comparison of S. agalactiae isolates from the same herd revealed little variability in their SmaI restriction patterns indicating a single strain originating from a common source of infection. However, comparison of S. agalactiae isolates between herds showed that each herd was infected with a distinct strain. The restriction profiles from S. dysgalactiae subsp. dysgalactiae and S. uberis were more diverse and often multiple strains of both species were present within an individual herd, with some herds containing more than one representative of a particular strain. Interestingly, it was only observed with S. dysgalactiae subsp. dysgalactiae that some strains were present in more than one herd. PFGE was found to be a useful and reproducible method for the discrimination of different strains of the three most important species of streptococci responsible for bovine mastitis and to offer a means to identify environmental sources of infection.