Staphylococcus spp. is a major cause of nosocomial infection and sepsis. However, increasing drug resistance is becoming a challenge to microbiologists. The purpose of this study was to identify and determine antimicrobial resistance phenotypes and drug resistance genes of clinical coagulase-negative staphylococci (CoNS) isolates at Mae Sot Hospital in Tak province, Thailand. A total of 229 CoNS isolates were collected from clinical specimens during two periods in 2014 and in 2015. Staphylococcus haemolyticus was the most prevalent species (37.55%), followed by S. epidermidis (21.83%), S. saprophyticus (11.79%) and S. hominis (11.35%) respectively. The remaining 17.48% of the organisms comprised S. capitis, S. arlettae, S. cohnii, S. equorum, S. xylosus, S. warneri, S. sciuri, S. pettenkoferi, S. kloosii and S. lugdunensis. Methicillin-resistant CoNS (MRCoNS), containing the mecA gene, were detected in 145 of 229 isolates, mostly found in S. haemolyticus and S. epidermidis. In addition, the differentiation of their macrolide–lincosamide–streptogramin B (MLSB) resistance phenotypes was determined by the D-test and corresponding resistance genes. Among 125 erythromycin-resistant CoNS, the prevalence of constitutive type of MLSB, inducible clindamycin resistance and macrolide–streptogramin B resistance phenotypes were 72, 13.60 and 14.40% respectively. These phenotypes were expressed in 80% of MRCoNS strains. In addition, the ermC gene (79.20%) was found to be more prevalent than the ermA gene (22.40%), especially among MRCoNS. These results indicate that CoNS may play an important role in spreading of drug resistance genes. More attention to these organisms in surveillance and monitoring programs is needed.