Objective: Germline pathogenic variants (PVs) in the DNA mismatch repair (MMR) genes and the base excision repair genes NTHL1 and MUTYH underlie hereditary CRC and polyposis syndromes. We evaluate the robustness and discriminatory potential of tumour mutational signatures in colorectal cancers (CRCs) for identifying germline PV carriers. Design: Whole exome sequencing of FFPE CRC tissue was performed on hereditary CRC PV carriers (14 MMR, 6 biallelic MUTYH, and 1 biallelic NTHL1), 9 sporadic MMR-deficient CRCs (MMRd controls) and 18 sporadic MMR-proficient CRCs (MMRp controls). COSMIC V3 Single Base Substitution (SBS) and Indel (ID) mutational signatures were calculated and assessed for their ability to differentiate CRCs that developed in carriers and non-carriers. Results: The combination of SBS18 and SBS36 contributing >23% of the signature profile was able to discriminate biallelic MUTYH carriers from MMRp and MMRd control CRCs with >99% confidence. Variant specific signatures SBS18 and SBS36 were identified for MUTYH p.Gly396Asp (p=0.015) and MUTYH p.Tyr179Cys (p=0.0012), respectively. SBS30 was significantly increased in a CRC from a biallelic NTHL1 carrier compared with MMRp and MMRd control CRCs. The combination of ID2, ID7, SBS15 and SBS1 could discriminate the 14 MMR PV carrier CRCs from the MMRp control CRCs, however, SBS and ID signatures, alone or in combination, could not provide complete discrimination between CRCs from MMR PV carriers and sporadic MMRd controls. Conclusion: Assessment of SBS and ID signatures can discriminate CRCs from MUTYH, NTHL1 and MMR PV carriers from non-carriers, demonstrating utility as a potential diagnostic and variant classification tool.