A total of 450 isolates of Staphylococcus aureus were typed using multilocus sequence typing (MLST). They were isolated from the cattle and environment of a UK organic dairy farm, 43 dairy herds in the USA, the mammary glands of cattle in Chile and the UK, and also included the reference strain Newbould 305 (NCIMB 702892). Strains were compared to investigate differences between isolates from varying sites of isolation and the population detected on a single farm. MLST was suitable for the differentiation of bovine associated S. aureus, and thirty different sequence types were detected. These contained a number of novel alleles detected at each loci, agreeing with previous reports of host-specialisation, and indicating localised strain evolution. MLST was also able to discriminate between isolates detected in milk and on teat skin. The N ewbould 305 strain was significantly (p<0.05) associated with teat skin, and this may have important implications for future studies. The majority of isolates (87.4%) were present within one previously undescribed clonal complex (CC97), which contained representatives from all three geographic locations. Analysis of isolates from a single farrri demonstrated the clonality of the organism, supporting the theory of cow-to-cow spread of disease. No isolates were detected in heifer mammary secretions, suggesting transmission to these animals did not occur. Typing of multiple colonies from a single sample demonstrated strain heterogeneity within individual quarters. Environmental isolates were detected, though it is unlikely that they formed a significant reservoir of infection on the farm studied.