Background: Microsatellite instability (MSI) is an established biomarker affecting immunotherapy outcomes and may influence DNA damage responses following chemoradiotherapy (CRT). While clinical studies have reported favorable CRT outcomes in MSI-high (MSI-H) colorectal tumors, the biological basis for differential radiosensitivity remains incompletely understood. To address this gap, we integrated clinical-radiomic analyses with functional assays using 3D organoid models derived from MSI-H and microsatellite-stable (MSS) colorectal cancer (CRC) cell lines. Methods: Clinical data from 87 rectal cancer patients receiving neoadjuvant CRT were analyzed. MSI status was determined by PCR and immunohistochemistry for MLH1, MSH2, MSH6, and PMS2. Radiomic features were extracted from pre-CRT CT images, and a machine learning model was trained to classify MSI status. For biological validation, 4 MSI-H (HCT116, HCT15, RKO, DLD-1) and 5 MSS cell lines (HT29, CACO2, SW480, COLO205, COLO320DM) were subjected to graded irradiation. Cells were embedded in 70% Matrigel or BME to generate 3D organoids. Radiation responses were evaluated using Calcein-AM-based viability area analysis, ATP-based 3D CellTiter-Glo assays, and area-under-curve (AUC) quantification across 0-8 Gy. Results: MSI tumors were exclusively found in the responder group (18.52%) and absent in non-responders (P<0.05). Pathological downstaging (T0-T2) was significantly higher in responders (88.89% vs. 38.33%, P<0.001), with increased nodal clearance (N0; 92.59% vs. 61.67%, P=0.003). In 3D organoid models, MSI-H cell lines displayed markedly greater radiosensitivity than MSS lines. MSI-H organoids showed stronger growth inhibition and lower viability in both Calcein-AM and ATP 3D assays, resulting in reduced radiation-response AUC values. In contrast, MSS organoids maintained higher viability and metabolic activity after irradiation, indicating relative radioresistance. Conclusion: MSI-H colorectal cancer demonstrated consistently higher chemoradiotherapy sensitivity across clinical outcomes, radiomic signatures, and 3D organoid-based functional assays. The enhanced radiosensitivity observed in MSI-H organoids suggests that deficiencies in DNA mismatch repair may contribute to increased susceptibility to radiation-induced damage, although the precise molecular pathways have not yet been fully defined. These integrated findings support the potential value of combining MSI testing with organoid-based functional profiling to refine patient stratification and guide personalized CRT approaches in colorectal cancer. Validation in larger and ethnically diverse cohorts will be necessary to confirm generalizability.