The present study aimed evaluate an on-farm culture system for identification of milk pathogens associated with clinical mastitis in dairy cows using two different gold standard approaches: standard laboratory culture in study 1 and 16S rRNA sequencing in study 2. In study 1, milk from mastitic quarters (i.e. presence of flakes, clots, or serous milk; n = 538) was cultured on-farm using a single plate containing three selective chromogenic media (Accumast-FERA Animal Health LCC, Ithaca, NY) and in a reference laboratory using standard culture methods, which was considered the gold standard. In study 2, mastitic milk was cultured on-farm and analyzed through 16S rRNA sequencing (n = 214). In both studies, plates were cultured aerobically at 37° C for 24 h and read by a single technician masked to gold standard results. Accuracy, sensitivity, specificity, positive (PPV) and negative predictive value (NPV) were calculated based on standard laboratory culture in study 1, and PPV was calculated based on sequencing results in study 2. Overall accuracy of Accumast was 84.9%. Likewise, accuracy for identification of Gram-negative bacteria, Staphylococcus sp., and Streptococcus sp. was 96.4%, 93.8%, and 91.5%, respectively. Sensitivity, specificity, PPV, and NPV were 75.0%, 97.9%, 79.6%, and 97.3% for identification of E. coli, 100.0%, 99.8%, 87.5%, and 100.0% for S. aureus, 70.0%, 95.0%, 45.7%, and 98.1% for other Staphylococcus sp., and 90.0%, 92.9%, 91.8%, and 91.2% for Streptococcus sp. In study 2, Accumast PPV was 96.7% for E. coli, 100.0% for Enterococcus sp., 100.0% for Other Gram-negatives, 88.2% for Staphylococcus sp., and 95.0% for Streptococcus sp., respectively. In conclusion, Accumast is a unique approach for on-farm identification pathogens associated with mastitis, presenting overall sensitivity and specificity of 82.3% and 89.9% respectively.