Control of the bovine mastitis pathogen Streptococcus uberis requires sensitive and epidemiologically meaningful subtyping methods that can provide insight into this pathogen's epidemiology and evolution. Development of a multilocus sequence typing (MLST) scheme based on six housekeeping and virulence genes allowed differentiation of 40 sequence types among 50 S. uberis isolates from the United States (n = 30) and The Netherlands (n = 20). MLST was more discriminatory than EcoRI or PvuII ribotyping and provided subtype data with better epidemiological relevance, e.g., by discriminating isolates with identical ribotypes obtained from different farms. Phylogenetic analyses of MLST data revealed indications of reticulate evolution between genes, preventing construction of a core phylogeny based on concatenated DNA sequences. However, all individual gene phylogenies clearly identified a distinct pauA-negative subtaxon of S. uberis for which housekeeping alleles closely resembled those of Streptococcus parauberis. While the average GC content for five genes characterized was between 0.38 and 0.40, pauA showed a considerably lower GC content (0.34), suggesting acquisition through horizontal transfer. pauA also showed a higher non synonymous/synonymous rate ratio (d(N)/d(S)) (1.2) compared to the other genes sequenced (d(N)/d(S) < 0.12), indicating positive selection in this virulence gene. In conclusion, our data show that (i) MLST provides for highly discriminatory and epidemiologically relevant subtyping of S. uberis; (ii) S. uberis has a recombinatorial population structure; (iii) phylogenetic analysis of MLST data reveals an S. uberis subtaxon resembling S. parauberis; and (iv) horizontal gene transfer and positive selection contribute to evolution of certain S. uberis genes, such as the virulence gene pauA.