Restriction enzyme fragmentation pattern (REFP) analysis was used to recognise Staphylococcus aureus strain variation in naturally occurring bovine subclinical mastitis. Multiple colony REFP analysis identified eight distinct strains of S. aureus in addition to the original strains A and B that were infused via the intramammary route, indicating that individual quarters of the udder may be colonised simultaneously by more than one strain of S. aureus. Examination of multiple colonies per milk sample may benefit bacterial strain recognition as an epidemiological tool in mastitis investigations. The dynamics of intramammary infection were determined using a novel double crossover experimental challenge. Quarters remained persistently infected for several weeks following challenge in all four cows, irrespective of the challenge strain. This indicated that no alteration of the original subclinical infection, including the possible induction of clearance of the quarters infected with S. aureus, or replacement of the original strain by the infused strain was induced by challenge. The persistent subclinical infection in all four animals supports previous reports on the chronicity of S. aureus intramammary infection in dairy cows.