A case of Staphylococcus aureus intramammary infection (IMI) on a Japanese dairy farm was monitored for 9 months. S. aureus isolates from cows and environmental samples consisted of specific strains with sequence types 352 and 705, as determined by multilocus sequence typing. Clonal strains of these sequence types are isolated from cows worldwide, indicating that they are adapted to the bovine environment. These results explain why many IMI cases are persistent and lead to subclinical mastitis. The strain isolated from milk was identical to those isolated from the cows' bodies and cows carrying S. aureus, milking units, personnel, heifers and cats in the dairy barn. These locations and factors should be emphasized as sources and routes of strains causing IMI.