The present work is a large epidemiological study aiming to detect the prevalence of subclinical mastitis and to investigate the major udder pathogens in Jalisco State, western Mexico. For this purpose, 2205 dairy cows, representing 33 Mexican dairy herds, were involved. Of 2205 cows, 752 mastitic animals were diagnosed and only 2,979 milk samples could be obtained for further investigation. All 2979 milk samples were subjected to California Mastitis Test (CMT) to differentiate clinical cases from subclinical ones where 1996 samples (67 %) reacted positively. Of these, 1087 samples (54.5%) came from cows suffering from clinical cases of mastitis. Bacteriological identification of the causative agents revealed the presence of a major group of pathogens including the Coagulase negative staphylococci (CNS), S.aureus, S.agalactiae, Corynebacterium spp. and Coliform bacteria which were detected in 464 (15.6%), 175 (5.9%), 200 (6.8%), 417 (14%) and 123 (4.1%) of the 2927 investigated quarters, 295 (15.4%), 118 (15.7%), 111 (14.8%), 227 (30.2%) and 109 (14.5%) of the 752 examined cows and in 33 (100%), 22 (66.7%), 19 (57.6%), 30 (90.1%) and 27 (81.8%) of the 33 herds involved, respectively. Other pathogens could be detected in the investigated milk samples such as S. dysgalactiae (0.4%), S.uberis (0.37%), Bacillus spp. (1%), Nocardia spp. (0.6%) und Candida spp. (0.1%). Meanwhile, others were present in a negligible ratio; including the Aerococcus viridans, and Enterococcus spp., Lactococcus lactis, S. bovis.