Genotypic and phenotypic analysis of 42 strains of Streptococcus uberis isolated from mammary secretions of 17 cows collected at different periods of the lactation cycle and from episodes of clinical mastitis were performed. Seventeen restriction endonuclease fingerprint (REF) patterns and 12 bacteriocin-like inhibitory substance (BLIS) fingerprints were observed. REF identified and differentiated closely related strains of S. uberis isolated from mammary secretions collected from the same cow at different periods of the lactation cycle and from episodes of clinical mastitis. BLIS fingerprinting of S. uberis complemented REF results. REF and BLIS fingerprinting provided evidence concerning persistence of infection in the same quarter or different quarters of the mammary gland over different periods of the lactation cycle, and occurrence of infection with similar and dissimilar strains of S. uberis. Biochemical profiles could not identify closely related strains nor did they complement REF results. Antibiotic resistance patterns alone were of little value in differentiating closely related strains, but were identical with isolates having same REF pattern. None of the S. uberis strains was found to carry plasmids. REF and BLIS fingerprinting can be utilized effectively to differentiate closely related and unrelated strains of S. uberis isolated from bovine mammary secretions.