Subclinical mastitis (SCM) is one of the most prevalent diseases affecting dairy animals and hindering the development of animal production sector worldwide. Staphylococci are the most significant causative bacterial pathogens in both clinical and subclinical cases. The present study aimed to investigate the prevalence of SCM among sheep detecting the prevalence of coagulase negative staphylococci (CNS) and studying some of their phenotypic and genotypic characters. A total of 145 individual half milk samples (HMSs) were collected aseptically from 75 apparently healthy ewes and examined. The prevalence of SCM based on California Mastitis Test (CMT) was 29.3 and 21.4% at sheep and udder HMSs levels, respectively. The prevalence of CNS in subclinically mastitic sheep was investigated in 31 (25.8%) HMSs. Identification of CNS isolates revealed that, S. epidermidis was the most prevalent (37.5%) followed by S. xylosus (25%) and each of S. simulans, S. chromogenes and S. haemolyticus (12.5%). The results of in-vitro antimicrobial susceptibility of CNS isolates against 12 antimicrobial agents showed high resistance against ampicillin, amoxicillin-clavulanic acid, cefoxitin and cefotaxime. Meanwhile, high susceptibilities were recorded against ciprofloxacin, levofloxacin, florophenicol, vancomycin, doxycycline, clindamycin, gentamicin and sulfamethoxazole-trimethoprim. The haemolytic activity and biofilm formation on CRA medium were investigated in all isolates. The haemolytic activity was detected in 75% of isolates meanwhile 62.5% of isolates were biofilm formers. The results of genotypic detection of mecA and blaZ resistance genes and icaD biofilm coding gene using PCR showed that they were detected in 80, 60 and 60% of the tested isolates, respectively. It was concluded that CNS isolates were the most prevalent causes of ovine SCM and the existence of high percentages of antimicrobials resistance as well as resistance and virulence genes represent risk factors and public health hazards and possible danger of lateral transfer of resistance genes to other microorganisms in both animals and humans.