Mutations in the DNA mismatch repair system increase mutation and recombination. They may thereby promote the genetic divergence that underlies speciation, after which the reacquisition of a functional repair system may sustain that divergence by creating a barrier to recombination. We tested several lines of Escherichia coli, derived from a common ancestor and evolved for 20,000 generations, for their recombination ability. Some lines, but not others, had become mismatch repair-defective mutators during experimental evolution, providing different opportunities for DNA sequence divergence. We knocked out the repair system in lines that had retained this function, and we restored function to those lines that had become defective. We then estimated recombination rates in various crosses between these repair-deficient and -proficient strains. The effect of the mismatch repair system on recombination was greatest in those lines that had evolved nonfunctional repair, indicating they had undergone more sequence divergence and, consequently, were more sensitive to the recombination-inhibiting effect of a functional repair system. These results demonstrate the establishment of an incipient genetic barrier between formerly identical lines, and they support a model in which the mismatch repair system can influence speciation dynamics through its simultaneous effects on mutation and recombination.