Klebsiella pneumoniae (K. pneumonia, KpI) is a predominate inducement of bovine mastitis, which is associated with high mortality and milk yield reduction. However, data is lacking on the molecular characteristics of bovine K. pneumoniae, limiting the risk assessment of its transmission through the food chain. Herein, we investigated the prevalence of K. pneumoniae in 6301 clinical mastitis (CM) milk samples from dairy cattle in northern area of China. In total, 183 K. pneumoniae isolates were recovered, with detection rates of 3.0% and 2.8% in 2018 and 2019, respectively. Like human clinical K. pneumoniae, all CM K. pneumoniae isolates belonged to one of three phylogroups: KpI (n = 143), Klebsiella quasipneumoniae subsp. similipneumoniae (KpII-B) (n = 37), and Klebsiella variicola (KpIII) (n = 3). We detected the extendedspectrum β-lactamase-encoding genes blaSHV-2a, blaCTX-M-14, and blaCTX-M-15, as well as clpC, lpfA, lacI, lacZ, lacY, and the fecABDEIR operon in the KpI isolates, which may contribute to their pathogenicity and host adaptability in cows. The high prevalence of KpI in dairy farms may be problematic, as it showed relatively higher rates of antibiotic resistance and virulence gene carriage than the KpII-B and KpIII isolates. Furthermore, we observed distinct differences in population structure between CM- and human infection-associated KpI isolates, with the genes associated with invasive infection in humans rarely being observed in bovine isolates, indicating that few CM-associated K. pneumoniae isolates pose a threat to human health. Nevertheless, bovine KpII-B isolates shared a high level of nucleotide sequence identity with isolates from human infections and frequently carried the nitrogen-fixation gene nif, suggesting an association between KpII-B isolates from cattle and humans, and plant-derived bacteria.