Background: Abnormal DDR is a hallmark of cancer, relating to genome instability, anti-tumor immunity, and sensitivity to chemotherapeutic agents and radiation. We conducted a large-scale investigation to clarify the alteration of DDR pathway in CRC. Methods: Tumor samples from 9321 CRC patients were retrospectively reviewed. Next-Generation Sequencing (NGS) on a custom-designed panel enriching 592 gene targets was performed. Samples with mutations detected in any of 29 DDR-related genes were deemed DDR-mutant (DDR-MT); the rest DDR-wild type (DDR-WT). Microsatellite instability (MSI) status was tested with a combination of immunohistochemistry (IHC), fragment analysis and NGS. Tumor mutational burden (TMB) was calculated based on somatic nonsynonymous missense mutations. PD-L1 was tested by IHC (SP142). Consensus molecular subtype (CMS) was developed using RNA sequencing data. Results: Of 9321 cases, 1290 (13.8%) were DDR-MT. DDR-MT frequency was higher in right vs. left sided (20.9% vs 10.8%, p < 0.001) and MSI-H vs. MSS (76.4% vs 9.5%, p < 0.001) cases. In the MSS cases, right-sided had marginally higher frequency of DDR-MT than left-sided (10.6% vs 9.1%, p = 0.055), with much higher frequency of Fanconi anemia pathway alteration in right-sided (1.5% vs 0.7%, p < 0.01). CMS1 subtype had the highest frequency of DDR-MT (34.8%); CMS2 had the lowest (7.1%). DDR-MT cases (vs. DDR-WT) had higher mutation rate of ARID1A (55.0% vs 19.1%, p < 0.0001), PIK3CA (22.6% vs 15.8%, p < 0.0001) and BRAF (20.4% vs 7.3%, p < 0.0001), and lower mutation rate of TP53 (48.2% vs 76.1%, p < 0.0001), APC (60.5% vs 74.5%, p < 0.0001) and KRAS (44.0% vs 49.8%, p < 0.001). Mean TMB was much greater in DDR-MT than DDR-WT (All: 20.9/Mb vs 7.7/Mb, p < 0.0001; MSS: 13.7/Mb vs 7.6/Mb, p < 0.05). PD-L1 positivity was also higher in DDR-MT compared to DDR-WT (All: 10.1% vs 2.7%, p < 0.0001; MSS: 4.8% vs 2.4%, p < 0.0001). Conclusions: Alteration of the DDR pathway was strongly associated with MSI status in CRC. The primary tumor sidedness might also be related, as DDR-MT was more prevalent in right-sided tumors. Elevated TMB and PD-L1 expression in DDR-MT CRC indicate more activated anti-tumor immune profiles compared to DDR-WT, regardless of MSI status, suggesting possible therapeutic benefit from immune checkpoint inhibitors in DDR-MT CRC.