The eukaryotic DNA mismatch repair (MMR) system contributes to maintaining genome integrity and DNA sequence fidelity in at least two important ways: by correcting errors arising during DNA replication, and also by preventing recombination events between divergent sequences. This study aimed to investigate the role of one key MMR gene in recombination. We obtained a mutant line in which the AtMLH1 gene has been disrupted by the insertion of a T-DNA within the coding region. Transcript analysis indicated that no full-length transcript was produced in mutant plants. The loss of a functional AtMLH1 gene led to a significant reduction in fertility in both homozygotes and heterozygotes, and we observed a strong bias against transmission of the mutant allele. To investigate the role of AtMLH1 in mitotic recombination, the mutant was crossed to a series of recombination reporter lines. A strong decrease (72%) in the frequency of homologous recombination was observed in the mutant. However, the decline in recombination due to homeology was less severe in the Atmlh1 mutant than in a wild-type control. These data demonstrate a dual role for AtMLH1 in recombination: it is both required for recombination and acts to limit recombination between diverged sequences.