A major shortcoming to plasmid-based genetic tools is the necessity of using antibiotics to ensure plasmid maintenance. While selectable markers are very powerful, their use is not always practical, such as during in vivo models of bacterial infection. During previous studies, it was noted that the uncharacterized LAC-p01 plasmid in Staphylococcus aureus USA300 isolates was stable in the absence of a known selection and therefore could serve as a platform for new genetic tools for Staphylococcus species. LAC-p01 was genetically manipulated into an Escherichia coli-S. aureus shuttle vector that remained stable for at least 100 generations without antibiotic selection. The double- and single-stranded (dso and sso) origins were identified and found to be essential for plasmid replication and maintenance, respectively. In contrast, deletion analyses revealed that none of the four LAC-p01 predicted open reading frames were necessary for stability. Subsequent to this, the shuttle vector was used as a platform to generate two plasmids. The first plasmid, pKK22, contains all genes native to the plasmid for use in S. aureus USA300 strains, while the second, pKK30, lacks the four predicted open reading frames for use in non-USA300 isolates. pKK30 was also determined to be stable in Staphylococcus epidermidis Moreover, pKK22 was maintained for 7 days postinoculation during a murine model of S. aureus systemic infection and successfully complemented an hla mutant in a dermonecrosis model. These plasmids that eliminate the need for antibiotics during both in vitro and in vivo experiments are powerful new tools for studies of StaphylococcusIMPORTANCE Plasmid stability has been problematic in bacterial studies, and historically antibiotics have been used to ensure plasmid maintenance. This has been a major limitation during in vivo studies, where providing antibiotics for plasmid maintenance is difficult and has confounding effects. Here, we have utilized the naturally occurring plasmid LAC-p01 from an S. aureus USA300 strain to construct stable plasmids that obviate antibiotic usage. These newly modified plasmids retain stability over a multitude of generations in vitro and in vivo without antibiotic selection. With these plasmids, studies requiring genetic complementation, protein expression, or genetic reporter systems would not only overcome the burden of antibiotic usage but also eliminate the side effects of these antibiotics. Thus, our plasmids can be used as a powerful genetic tool for studies of Staphylococcus species.