Polymerase chain reaction-based DNA fingerprinting was used as a tool to differentiate new and persistent Streptococcus uberis and Streptococcus dysgalactiae intramammary infections (IMI) in dairy cows. The same subtype of S. uberis or S. dysgalactiae was detected from some infected mammary glands from one lactation to the next documenting the persistence of these infections. Conversely, some streptococci isolated from mammary glands during a lactation or from one lactation to the next were different subtypes suggesting that a new IMI occurred. These new streptococcal IMI would never have been detected using phenotypic methods of streptococcal identification. Results of this study indicate that PCR-based DNA fingerprinting can be used as an effective procedure to differentiate new and persistent S. uberis and S. dysgalactiae IMI in dairy cows. This technique will be useful in epidemiological investigations, and drug and vaccine efficacy studies when attempting to delineate new and persistent IMI.