Culture-independent PCR-DGGE fingerprinting was used to reveal the bacterial composition and diversity associated with raw milk of mastitis cows from Hokkaido, Japan for the first time. All the mastitis milk samples were diagnosed as solely infection by Coliforms using the classical microbiological method based on on-farm culturing. Our results revealed that the bovine mastitis-associated bacteria were host-specific because community structure varied between each sample. Klebsiella pseudomoniae, Lactococcus lactis Staphylococcus aureus and Escherichia sp were found to be the widely distributed species. Furthermore, more than one mastitis-causing pathogen was found to be present in some mastitis samples. These pathogens may not only act as etiology agents but also play a role in disrupting the natural microbial ecology in mastitis bovine. This finding highlights the limitation of the traditional identification and characterization strategy. Therefore, it is suggested that the methodology applied in this study might be a valuable addition to mastitis control and prevention.