The lp28-1 plasmid is required for persistent infection by the Lyme disease spirochete, Borrelia burgdorferi. Mutational studies on this plasmid have shown that the vls locus is important for antigenic variation of the VlsE lipoprotein that leads to immune evasion and persistence. However, it is still unknown whether the vls system is the only genetic locus on this plasmid necessary for long-term infection, and thus the potential role of non-vls genes on lp28-1 in virulence and persistence is yet to be fully determined. Despite extensive mutational analyses, two lp28-1 regions containing the ORFs bbf19 - bbf22 and bbf27 – bbf30 have not yet been mutated in their entirety. In this study, we set out to establish if these unstudied regions of lp28-1 play a role in spirochete persistence. Results show that the generated mutants were fully infectious in immunocompetent mice, and were able to persist for 91 days following infection. Following this finding, ospC expression by these mutants was determined, as it has been reported that spirochetes lacking lp28-1 fail to downregulate expression of this lipoprotein leading to immune clearance. Data presented here failed to show a definitive difference in ospC expression levels during host infection when the mutants were compared to the wild type. Overall, the results strongly suggest that non-vls genes residing on lp28-1 do not play a role in spirochete persistence during infection of the mammalian host, and that the regions under study are likely not involved in the regulation of ospC expression. In conjunction with previous studies involving mutation of non-vls loci on lp28-1, these findings suggest that the vls locus is likely the sole genetic element on this plasmid responsible for immune evasion and persistence exhibited by the Lyme disease pathogen.