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DNA mismatch repair (MMR) corrects replication errors and is recruited by the histone mark H3K36me3, enriched in exons of transcriptionally active genes. To dissect in vivo the mutational landscape shaped by these processes, we employed single-cell exome sequencing on T cells of wild-type and MMR-deficient (Mlh1-/-) mice. Within active genes, we uncovered a spatial bias in MMR efficiency 3' exons, often H3K36me3-enriched, acquire significantly fewer MMR-dependent mutations compared with 5' exons. Huwe1 and Mcm7 genes, both act