Until recently, few prognostic signatures for colorectal cancer (CRC) patients receiving 5-fluorouracil (5-FU)-based chemotherapy could be used in clinical practice. Here, using transcriptional profiles for a panel of cancer cell lines and three cohorts of CRC patients, we developed a prognostic signature based on within-sample relative expression orderings (REOs) of six gene pairs for stage II-III CRC patients receiving 5-FU-based chemotherapy. This REO-based signature had the unique advantage of being insensitive to experimental batch effects and free of the impractical data normalization requirement. After stratifying 184 CRC samples with multi-omics data from The Cancer Genome Atlas into two prognostic groups using the REO-based signature, we further revealed that patients with high recurrence risk were characterized by frequent gene copy number aberrations reducing 5-FU efficacy and DNA methylation aberrations inducing distinct transcriptional alternations to confer 5-FU resistance. In contrast, patients with low recurrence risk exhibited deficient mismatch repair and carried frequent gene mutations suppressing cell adhesion. These results reveal the multi-omics landscapes determining prognoses of stage II-III CRC patients receiving 5-FU-based chemotherapy.