Fifty plasmid-free strains of Streptococcus uberis, including 40 strains isolated from cows in two dairy herds in Tennessee with subclinical and clinical mastitis, 9 strains isolated from cows in the US, and 1 strain from Great Britain were examined for genomic DNA restriction fragment length polymorphisms. The 50 strains belonged to 35 DNA fingerprint patterns. Within a herd, fingerprint patterns from isolates obtained from cows with subclinical mastitis at different periods of the lactation cycle and from episodes of clinical mastitis were similar. Restriction fragment length polymorphism analysis revealed the presence of DNA fragments > or = 21 kb in all 12 strains of S. uberis isolated from episodes of clinical mastitis and in only 8 of the 38 (21%) remaining strains. Cluster analysis placed the 50 strains into eight distinct clusters. Most strains from the northeastern US were confined to one cluster, whereas most of the isolates from episodes of clinical mastitis belonged to two different clusters. These data suggest that clonal diversity exists among strains of S. uberis isolated from bovine mammary secretions.