Streptococcus uberis is an important bovine mastitis pathogen, but not all isolates have equal capacity to cause disease. The aims of this study were to identify possible virulence-associated genes that could be used to identify isolates with enhanced virulence. DNA from a pool of putative commensals was subtracted from a clinical pool resulting in a set of DNA sequences (probes) that were enriched in the clinical mastitis group. The probes were hybridised with DNA from a collection 29 isolates from cases of clinical mastitis and isolates not associated with disease. Hybridization revealed five major clusters. The first cluster (7 isolates) consisted almost entirely of commensals, while the second (7 isolates) was mixed. The remaining three clusters contained 15 S. uberis isolates from cows with clinical mastitis. Twenty-six probes were selected for sequencing based on principal component analysis (PCA) or their presence mainly in clinical isolates. PCA identified five probes with clear differences in intensity between signals from clinical isolates and commensals; these probes could represent novel virulence determinants. Manual inspection of arrays identified genes prominent among clinical isolates that specify carbohydrate and lipid metabolism (possible role in the growth or survival of S. uberis in milk) and genes specifying hypothetical proteins, possibly novel virulence factors. The common occurrence, among clinical isolates, of probes having homology with transposases and insertion sequences suggests recent acquisition of factors that could be associated with virulence. These results suggest the existence of a subset of S. uberis with enhanced virulence, due possession of virulence-associated gene sequences.