In the canonical DNA mismatch repair (MMR) mechanism in bacteria, if during replication a nucleotide is incorrectly mis-paired with the template strand, the resulting repair of this mis-pair can result in the degradation and re-synthesis of hundreds or thousands of nucleotides on the newly-replicated strand (long-patch repair). While mycobacteria, which include important pathogens such as Mycobacterium tuberculosis, lack the otherwise highly-conserved enzymes required for the canonical MMR reaction, it was found that disruption of a mycobacterial mismatch-sensitive endonuclease NucS results in a hyper-mutative phenotype, which has led to the idea that NucS might be involved in a cryptic, independently-evolved DNA MMR mechanism. It has been proposed that nuclease activity at a mismatch might result in correction by homologous recombination (HR) with a sister chromatid. Using oligonucleotide recombination, which allows us to introduce mismatches during replication specifically into the genomes of a model for M. tuberculosis, Mycobacterium smegmatis, we find that NucS participates in a direct repair of DNA mismatches where the patch of excised nucleotides is largely confined to within ~5 - 6 bp of the mis-paired nucleotides, which is inconsistent with mechanistic models of canonical mycobacterial HR or other double-strand break (DSB) repair reactions. The results presented provide evidence of a novel NucS-associated mycobacterial MMR mechanism occurring in vivo to regulate genetic mutations in mycobacteria.