Abstract: Objective: To investigate the resistance mechanism and prevalence of linezolid-resistant coagulase-negative staphylococci in Jiangsu Province. Methods: A total of 38 strains of linazolid-resistant coagulase-negative staphylococci, including 32 strains of Staphylococcus capitis, 4 strains of Staphylococcus hominis, 1 strain of Staphylococcus epidermidis and 1 strain of Staphylococcus lentus, were collected from the clinical and environmental samples of three tertiary hospitals in Jiangsu Province during 2017 and 2018. The drug sensitivity of these strains were detected by the broth dilution method, and the minimal inhibitory concentration (MIC) of linezolid was detected by E-test. The genes in domain V of 23S rRNA, cfr and optrA genes were detected by PCR and sequencing technology. The homology analysis of these strains was performed with the pulsed field gel electrophoresis (PFGE). Results: All of 38 strains of coagulase-negative staphylococci were resistant to linezolid, penicillin and oxacillin, and were sensitive to vancomycin. The drug resistance rates to clindamycin, levofloxacin and ciprofloxacin were above 95%. Cfr gene was detected in 31 out of 38 strains. The G2576T mutation in domain V of 23S rRNA was detected in 35 strains, in which C2319T mutation was also detected in 2 strains of Staphylococcus hominis. In addition, C2319T mutation was also found in 1 strain of Staphylococcus hominis and 1 strain of Staphylococcus epidermidis. All strains were negative for optrA gene. PFGE showed that 32 strains of Staphylococcus capitis were highly similar and from the same clone, and that 4 strains of Staphylococcus hominis were also from the same clone. Conclusion: The prevalent linezolid-resistant coagulase-negative staphylococci in Jiangsu Province are mainly caused by cfr gene and gene mutations in domain V of 23S rRNA, and there are clonal spread of Staphylococcus capitis and Staphylococcus hominis.