This research was committed to evaluate the prevalence of bovine subclinical mastitis for isolation and identification of the microbial agents and predisposing factors associated with subclinical mastitis in randomly selected 250 lactating cows (179 local cattle and 71 (crossbreds) at Satkhira District of Bangladesh. California Mastitis Test was carried out each quarter sample of lactating dairy cows for evaluation of subclinical mastitis. Among 250 lactating cows, 66 (26.4%) were found to be positive for subclinical mastitis by California Mastitis Test. Out of 66 California Mastitis Test positive cows, only 19 cows showed strong positive reactions and these samples were used in culture media for isolation and identification of microbial agents. The prevalence of subclinical mastitis was significant in crossbreds (35.21%) as compared to local cattle (Bos indicus) (21.79%) . There was a dramatic shot up of prevalence in cows with a daily milk yield of 5-7 L and 7-10 L. The maximal prevalence found in mid-lactation was 30.99% and the greatest prevalence in the age group of 9 to 12 years was 37.5%. The prevalence of subclinical mastitis was comparatively superior (32.69%) in lactating cows were housed with fictile floor than that of the slatted floor (16.66%). Among the microorganisms isolated from the strongly positive subclinical mastitis samples, the most frequent microbes were Staphylococcus sp. (73.68%). For early detection of subclinical mastitis, California Mastitis Test can be accomplished regularly as a control measure and emphasis should be demonstrated on farm management practices, especially on milking equipments and hygienic udder. 1525837904.php