Improved methods for predicting chemoresponsiveness involving the identification of polymorphic markers is highly desirable, considering narrow therapeutic index and frequent resistance to anti-cancer regimens. The genome-wide screening of chemosensitive single nucleotide polymorphisms (SNPs) was undertaken in association with in vitro chemosensitivity assays in 104 colorectal cancer patients for the initial screening step. Allele frequency, linkage disequilibrium, potential function, and Hardy-Weinberg equilibrium of the candidate SNPs were then determined for the identifying step. Finally, clinical association analysis in the other 260 evaluable patients or cell viability assays of transfected RKO cells was used to verify candidate SNPs for the validation step. In total, 12 SNPs to six regimens were initially chosen during the screening and identifying steps. In patients receiving fluoropyrimidine-based adjuvant chemotherapy, the substitution alleles of GPC5 rs553717 (AA) correlated significantly with tumor recurrence and shorter disease-free survival (P = 0.019 and 0.023, respectively). Interestingly, RKO cells expressing mutant GPC5 showed enhanced cell death in response to 5-FU in cytotoxicity assays. Patients that were homozygous for the reference alleles SSTR4 rs2567608 (AA) and EPHA7 rs2278107 (TT) showed lower disease control rates in response to irinotecan and oxaliplatin regimens, respectively, than those with substitution alleles (P = 0.022 and 0.014, respectively). Thus, we identified chemosensitive SNP markers using a novel three step process of genome-wide analysis consisting of in vitro screening, identification, and validation. The candidate chemosensitive SNP markers identified in our study, including those identified in vitro, can now be further verified in a large cohort study.