Colorectal cancer (CRC) is the third leading cause of cancer-related deaths worldwide. Studies have shown that the DNA damage response (DDR) mutation is strongly associated with microsatellite instability (MSI) status and is an indication for patients with CRCs receiving immune checkpoint inhibitor (ICI) treatment. However, DDR mutation in microsatellite stable (MSS) CRC remains unclear.In this study, Fisher's exact test, Student'st-test, Wilcoxon rank-sum test and Cox proportional hazards regression model were performed, and a p value of < 0.05 was considered statistically significant.The most common gene alterations were APC (77%), TP53 (73%), KRAS (48%), and PIK3CA (25%). The mutationfrequency of APC and TP53 in left-sided CRC was significantly higher than that for right-sided CRC, while the mutation frequency of PIK3CA, ACVR2A, FAT4, and RNF43 in right-sided CRC was significantly higher than that for left-sided CRC. DDR mutations occurred in100% of MSI CRCs and in 83.77% of MSS CRCs, with the most frequently mutated DDR genes being ARID1A (7.5%), ATM (5.7%,) and BRCA2 (2.6%). When right- and left-sided CRCs were compared, no significant difference was observed for DDR genes and pathways. A survival analysis indicated that the DDR mutation was not associated with overall survival (OS) in MSS CRCs, while left-sided patients with homologous recombination repair (HRR) pathway mutations had a significantly prolonged OS compared with right-sided CRCs.Here, we found that stage and grade were statistically significant independent prognostic factors in the left-sided CRC and the right-sided CRC, recommending treatment for these patients stratified by stage. For the future, utilizing DDR gene defects for expanding treatment options and improving prognosis is an issue worth exploring.© 2023 The Authors. Cancer Medicine published by John Wiley & Sons Ltd.