AbstractCarriers of germline biallelic pathogenic variants in theMUTYHgene have a high risk of colorectal cancer. We test 5649 colorectal cancers to evaluate the discriminatory potential of a tumor mutational signature specific toMUTYHfor identifying biallelic carriers and classifying variants of uncertain clinical significance (VUS). Using a tumor and matched germline targeted multi-gene panel approach, our classifier identifies all biallelicMUTYHcarriers and all known non-carriers in an independent test set of 3019 colorectal cancers (accuracy = 100% (95% confidence interval 99.87–100%)). All monoallelicMUTYHcarriers are classified with the non-MUTYHcarriers. The classifier provides evidence for a pathogenic classification for two VUS and a benign classification for five VUS. Somatic hotspot mutationsKRASp.G12C andPIK3CAp.Q546K are associated with colorectal cancers from biallelicMUTYHcarriers compared with non-carriers (p = 2 × 10⁻²³andp = 6 × 10⁻¹¹, respectively). Here, we demonstrate the potential application of mutational signatures to tumor sequencing workflows to improve the identification of biallelicMUTYHcarriers.