Coagulase-negative staphylococci are mostly known as comensals on the body of humans and animals, however they are also important pathogens and often cause diseases in immunocompromised patients or patients with artificial medical devices as well as mastitis in cattle. Many strains are resistant to various antimicrobial agents and the resistance genes are often carried on mobile genetic elements such as plasmids, prophages and transposons, which can spread the resistance to other bacterial species. A detailed analysis of plasmids in coagulase-negative staphylococci from human and veterinary clinical material was performed. Plasmids were isolated by isolation kits and cleaved by restriction endonucleases to determine their size. Plasmids from different strains were compared with each other and with S. aureus plasmids. Correlation between plasmid content and antimicrobial resistance was analysed. Antimicrobial resistance genes were detected on plasmids using PCR. Plasmid curing was performed by sodium dodecyl sulfate to determine the role of plasmids in antimicrobial resistance. Using electroporation, plasmids shared by different staphylococcal species were transferred into cells after plasmid curing. The majority of analysed strains contained plasmids, the highest number of plasmids in one strain was five. Restriction analysis revealed identical plasmids in different staphylococcal species. Most analysed strains were resistant to at least one antibiotic, one strain was resistant up to five different antibiotics. Genes conferring resistance to tetracycline and erythromycin were characterized and their location on plasmids was confirmed by plasmid curing. S. petrasii strain after plasmid curing was transformed by identical plasmid from S. aureus, proving the possibility of horizontal transfer of plasmids among different staphylococcal species. This work confirmed that coagulase-negative staphylococci are resistant to a high number of antibiotics and the resistance genes can be located on plasmids. The presence of identical plasmids in different staphylococcal species and their transfer by electroporation indicates the horizontal transfer of plasmids between species and the importance of plasmids in spread of antimicrobial resistance. We kindly acknowledge the financial support from the research project granted by the Ministry of Agriculture of the Czech Republic (QJ1510216).