Streptococcus uberis is one of the principal causative agents of bovine mastitis. The organism is typically considered an environmental pathogen. In this study, two multilocus sequence typing (MLST) schemes and whole genome DNA microarrays were used to evaluate the degree and nature of genome flexibility between S. uberis strains. The 21 isolates examined in this study arise from a collection of 232 international isolates for which previous epidemiological and preliminary genotyping data existed. The microarray analysis resulted in an estimate of the core genome for S. uberis, consisting of 1530 ORFs, among 1855 tested, representing 82.5% of the S. uberis 0140J genome. The remaining ORFs were variable in gene content across the 21 tested strains. A total of 26 regions of difference (RDs), consisting of three or more contiguous ORFs, were identified among the variable genes. Core genes mainly encoded housekeeping functions, while the variable genes primarily fell within categories such as protection responses, degradation of small molecules, laterally acquired elements, and two component systems. Recombination detection procedures involving the MLST loci suggested S. uberis is a highly recombinant species, precluding accurate phylogenetic reconstructions involving these data. On the other hand, the microarray data did provide limited support for an association of gene content with strains found in multiple cows and/or multiple herds, suggesting the possibility of genes related to bovine transmissibility or host-adaptation. (C) 2008 Elsevier B.V. All rights reserved.