[Objective] In order to explore whether replication initiation protein (Rep) can be used as a molecular biomarker for the phylogenetic relationship study of natural plasmids. [Methods] The Reps of natural plasmids of Lactobacillus plantarum as a target, the phylogenetic relationships of these plasmids were analyzed and discussed in detail by constructing Rep phylogenetic tree. [Results] A total of 45 plasmids encoding Rep in Lactobacillus plantarum could be clustered into 5 closely related families and 1 single plasmid pG6302, of which family 1 to 4 plasmids could be further subdivided into 10 subfamily groups with closer evolutionary relationships, suggesting that these plasmids may originate from the 6 ancestral plasmids. [Conclusion] Since the amino acid sequences of Reps show suitable conservation and variability, it may be an ideal molecular biomarker for phylogenetic relationship study of plasmids encoding Rep in Lactobacillus plantarum. The results could provide a simple and effective method and standard, as well as the evidence and basis at the molecular level for phylogenetic evolution study of natural plasmids encoding Rep in Lactobacillus plantarum or other lactic acid bacteria.