Introduction: Coagulase negative Staphylococci are a part of normal human flora .They are an important cause of nosocomial infections worldwide. Previously they were considered as commensals but now they are considered as major cause of nosocomial bloodstream infections, urinary tract infections and various indwelling device‑related and prosthetic implants infections [3]. Aim: To determine the prevalence and susceptibility of Coagulase negative staphylococci and their antibiotic susceptibility testing from various clinical samples in a tertiary care centre. Materials & Methods: A cross sectional study was conducted in the department of microbiology, Saveetha medical college .76 strains of CONS were isolated from blood, urine and exudate from both OP and IP. Specimens were collected from various departments like ICU, NICU, General Medicine, SICU, MICU, OBG, ORTHO. Antibiotic susceptibility of all the isolated CONS was done according to CLSI guidelines. Result: In this study, most CONS species isolated were from blood (65%) followed by exudate (25%) and urine. Prevalence of CONS is more in females than in males among the people of 20-39 yrs of age. CONS were isolated more from GM and OBG wards. Staph epidermidis is the most prevalent CONS species followed by S.haemolyticus.26% were methicillin resistance CONS species. Regarding the antibiotic susceptibility, they showed high susceptibility to vancomycin, nitrofurantoin, linezolid, norfloxacin and resistance were seen in Penicillin, Cephalosporin group of drugs.