Staphylococcus aureus is a common cause of hospital, community and livestock-associated infections and is increasingly resistant to multiple antimicrobials. In S. aureus the majority of antimicrobial-resistance and virulence genes are carried on extrachromosomal plasmids. Only around 5% of sequenced S. aureus plasmids are considered self-transmissible conjugative plasmids, however 92% of non-conjugative plasmids are predicted to be mobilisable. The pWBG749 family of staphylococcal conjugative plasmids mobilize non-conjugative plasmids carrying mimics of the pWBG749 origin-of-transfer (oriT) sequence. These oriT mimics are carried by 53% of non-conjugative Staphylococcus aureus plasmids. The oriTs have diverged into five subtypes. Variants of the ribbon-helix-helix-domain protein SmpO, encoded by each conjugative plasmid, determine specificity for each oriT. Moreover, conjugative plasmids can mobilize non-cognate oriTs if the appropriate SmpO variant is present. Here we characterized four SmpO variants using surface-plasmon-resonance (SPR)-based DNA-binding assays, analytical gel filtration, small-angle X-ray scattering and other techniques. The SmpO proteins formed tetramers in solution and bound two DNA sites, IR2 and IR2*, located ~60 bp apart. SPR using single-nucleotide-substituted oligonucleotides delineated the critical nucleotides for three different SmpO-IR2* interactions. Each SmpO protein specifically bound a distinct IR2/IR2* motif, however, a single amino-acid substitution in SmpO enabled a switch in SmpO binding and oriT-mobilization specificity. Interestingly, two divergent conjugative plasmids, pWBG731 and pCO2, mobilized the same group of oriT sequences. This and other phylogenetic incongruences between conjugative plasmids and their oriT sequences were observed in multiple pWBG749-family lineages. Therefore, not only have conjugative plasmids diverged to carry distinct oriT sequences, but strangely, conjugative-plasmid oriT sequences have also seemingly converged—or have been replaced—with pre-existing oriT types on non-conjugative plasmids during evolution. We propose this apparent 'oriT switching' results from strong selection for mobilization of non-conjugative plasmids carrying an incompatible oriT. These observations suggest oriT specificity changes frequently during evolution and relaxase-in trans conjugative mobilization mechanism is likely a driving factor.