Decomposing somatic mutation spectra into mutational signatures and their corresponding etiologies provides a powerful approach for investigating the mechanism of DNA damage and repair. Assessing microsatellite (in)stability (MSI/MSS) status and interpreting their clinical relevance in different malignancies offers significant diagnostic and prognostic value. However, little is known about microsatellite (in)stability and its interactions with other DNA repair mechanisms such as homologous recombination (HR) in different cancer types. Based on whole-genome/exome mutational signature analysis, we showed HR deficiency (HRd) and mismatch repair deficiency (MMRd) occur in a significantly mutually exclusive manner in stomach and colorectal adenocarcinomas. ID11 signature with currently unknown etiology was prevalent in MSS tumors, co-occurred with HRd and was mutually exclusive with MMRd. Apolipoprotein B mRNA editing enzyme, Catalytic polypeptide-like (APOBEC) signature co-occurred with HRd and was mutually exclusive with MMRd in stomach tumors. The HRd signature in MSS tumors and the MMRd signature in MSI tumors were the first or second dominant signatures wherever detected. HRd may drive a distinct subgroup of MSS tumors and lead to poor clinical outcome. These analyses offer insight into mutational signatures in MSI and MMS tumors and reveal opportunities for improved clinical diagnosis and personalized treatment of MSS tumors.