Clostridium perfringens strains often carry multiple conjugative toxin or antibiotic resistance plasmids, most of which encode a similar Rep protein (97%-100% amino acid sequence identity), despite the conventional relationship between shared replication mechanisms and plasmid incompatibility. We have asked how these closely related plasmids are maintained within a single strain of C. perfringens. In many bacteria partitioning of plasmid DNA upon cell division involves a parMRC system. Phylogenetic analysis has highlighted the presence of at least ten different parMRC partitioning families (parMRCA-J) in C. perfringens, with differences in amino acid sequence identity between each ParM family ranging from 15% to 54%. Interestingly, no two plasmids encoding genes belonging to the same parMRC family were observed in a single strain, suggesting that these families represent the basis for plasmid incompatibility in C. perfringens. To validate the parMRC incompatibility groups, pairs of genetically marked plasmids encoding parMRC homologues were introduced into a single strain by conjugation. The relative stability of each plasmid was determined using an incompatibility assay in which the plasmid profile of each strain was monitored genetically over three days in the absence of direct selection. The results demonstrated that plasmids with identical parMRC homologues were incompatible and could not co-exist in the absence of external selection, whereas plasmids that had closely related, but different, parMRC homologues could co-exist in the same cell under these conditions. To further understand the mechanism by which these partitioning systems cause incompatibility, surface plasmon resonance was used to interrogate key recognition steps between ParR and parC components. Our results showed that ParR homologues interact with their cognate parC sequence more effectively than non-cognate parC sequence. In conclusion, these data begin to provide evidence that incompatibility of the conjugative toxin plasmids of C. perfringens is mediated by their parMRC-like partitioning systems.