To study molecular genetic characteristics and antibiotic resistance patterns of methicillin-resistance coagulase-negative staphylococci (CoNS) from mastitic cows in Egypt. Methods: The study material consisted of 205 milk samples. Milk was bacteriologically tested. Hemolytic activity and adhesive properties of CoNS isolates were also evaluated. Fifty CoNS isolates were submitted to in vitro susceptibility testing by the disk diffusion method. From the pool of 50 CoNS isolates, 15 Methicillin resistance isolates. were screened for genes encoding staphylococcal enterotoxin B (seb), toxic shock syndrome toxin1 (tss), intracellular adhesin D (icaD), and Methicillin resistance (mecA) by PCR. Results: CoNS were serotyped into three species S. haemolyticus, S. saprophyticus, and S. epidermidis. S. saprophyticus was the predominant CoNS species isolated from cows with clinical mastitis whereas S. epidermidis was the most prevalent one in subclinical mastitis cases. Susceptibility screening against 10 antibiotics determined 68% CoNS isolates as multidrug-resistant. High resistance was observed against penicillin (84%) and oxacillin (50%). The methicillin resistance gene, mecA was found in 73.33% isolates and interestingly, 26.67% of mecA negative isolates were oxacillin resistance. Of all the genes examined, 33.3% of examined isolates were positive for the staphylococcal enterotoxin B (seb) gene. Biofilm production was confirmed in 12% of CoNS. Conclusions: CoNS as an emerging cause for 60% of clinical mastitis and 67.27% of subclinical mastitis in cows in Egypt, and harboring the virulence genes rendering them as a potential threat for veterinary and public health and are also harboring resistance against penicillin and oxacillin the core for numerous novel intramammary combinations.