Capecitabine, an oral prodrug of 5-fluorouracil (5-FU), is part of the standard treatment of colorectal cancer (CRC). Severe adverse dose limiting reactions that impair treatment safety and lead to treatment suspension remain a relevant concern. Single-nucleotide polymorphisms (SNPs) in genes involved in the activation of capecitabine may alter the bioavailability of 5-FU and thereby affect therapy outcomes. The aim of this study was to evaluate the association of these SNPs with severe toxicity and treatment suspension in patients with CRC treated with capecitabine-based therapy. An ambispective cohort study was conducted, including 161 patients with CRC. SNPs were analyzed using real-time PCR with TaqMan probes. Toxicity was assessed according to the National Cancer Institute Common Terminology Criteria for Adverse Events v.5.0. CES1 rs71647871-A was associated with a severe hand-foot syndrome ( = 0.030; OR = 11.92; 95% CI = 1.46-73.47; GG vs. A). CDA rs1048977-CC ( = 0.030; OR = 2.30; 95% CI 1.09-5.00; T vs. CC) and capecitabine monotherapy ( = 0.003; OR = 3.13; 95% CI 1.49-6.81) were associated with treatment suspension due to toxicity. SNPs CES1 rs71647871 and CDA rs1048977 may act as potential predictive biomarkers of safety in patients with CRC under capecitabine-based adjuvant therapy.