The purposes of this study were to identify sources of Staphylococcus aureus on dairies and to determine whether S. aureus colonization of heifer body sites increases the risk of S. aureus IMI at parturition. In herds with high (> 10%) or low (< 3%) prevalence of S. aureus IMI, S. aureus was isolated from heifer teat skin, heifer external orifices, housing, feedstuffs, humans, nonbovine animals, air, and equipment. Additionally, in herds with high prevalence, S. aureus was isolated from bedding, insects, and water. The predominant sources of S. aureus for both groups were other IMI and heifer body sites. Heifers with prepartum lacteal secretions with S. aureus were at greater risk of S. aureus IMI at parturition than were prepartum heifers with lacteal secretions that were negative for S. aureus. Heifers with teat skin colonized by S. aureus were 3.34 times more likely to have S. aureus IMI at parturition than were noncolonized heifers. Overall, 35% of 700 heifers were colonized with S. aureus on a body site at least once. Although colonizations of most body sites appeared to be transient, a few heifers were colonized on the same site for 1 yr. Persistently colonized heifers may represent the primary reservoirs of S. aureus for other heifers.