<p>Bovine mastitis caused by <i>Klebsiella pneumoniae</i> poses significant economic and health challenges in dairy production, particularly in its subclinical form. In this study, milk samples (n = 305) from different cattle breeds of Red Sindhi, Sahiwal, Cholistani, and Holstein Friesian were collected from six dairy farms in Pakistan. The prevalence of subclinical mastitis ranged from 42.3% in Red Sindhi to 77.0% in Sahiwal breeds, with an overall prevalence of 56.39%. The prevalence of <i>K. pneumoniae</i> was 11.8% (36/305), varying significantly among breeds: Cholistani (26.2%), Red Sindhi (17.3%), Holstein Friesian (5.0%), and Sahiwal (0%). Farm-specific prevalence ranged from 0% to 26.2%, underscoring the influence of local management practices. All tested microorganisms remained sensitive to cefotaxime, meropenem, and ciprofloxacin but revealed 100% resistance to ampicillin and 80.5% and 69.4% resistance to amoxicillin and streptomycin, respectively. Molecular analysis detected the <i>bla</i>_TEM in 91.6% of isolates. These findings highlight significant breed and farm-level differences in subclinical mastitis and resistance patterns. The predominance of <i>bla</i>_TEM and full susceptibility to third-generation cephalosporins suggest that local <i>K. pneumoniae</i> isolates may differ from global trends. Strengthened farm management and prudent antimicrobial use are essential to limit infections and safeguard both animal and public health.</p> Graphical Abstract <p></p>