We have recently shown that low-passage, infectious Borrelia burgdorferi strain B31 MI is a heterogeneous mixture of clones varying in colony morphology, growth rate, protein profiles, plasmid content and infectivity. In this study, we asked whether there is a selection for certain clonal populations during the infectious cycle when uncloned B31 MI is used as the starting strain. B31 MI derivatives were reisolated from various tissues of two mice after completion of a mouse-tick-mouse infectious cycle and their protein and plasmid profiles were analyzed. Both mice developed ostensibly clonal infections despite the fact that the infectious cycle was started with a heterogeneous strain. Moreover, the mice became infected with two different clones varying in protein profile and growth phenotype. Comparison of the mouse reisolates to uncloned B31 MI and clonal variants derived from B31 MI before mouse-tick-mouse passage suggests that they were derived from clonal populations present in the uncloned B31 MI. Our results indicate the presence of at least two distinct populations within B31 MI that are competent to complete an experimental mouse-tick infectious cycle. The study provides insight into infectivity profiles and infection dynamics of different clonal populations present in a low-passage, infectious B. burgdorferi strain.