Background: Microsatellite instability (MSI) plays a crucial role as a cancer immunotherapy and prognosis biomarker, making accurate MSI detection essential. However, currently, there is still a lack of available data on the impact of neoadjuvant therapy on MSI status, as well as validated data on standardized methods for MSI testing in small biopsy samples. The study aimed to investigate the concordance of MSI status between paired biopsy and surgical samples, as well as the impact of neoadjuvant therapy on MSI status using a novel MSI next-generation sequencing (MSI-NGS) detection panel. Methods: A total of 137 colorectal cancer (CRC) patients were enrolled for this study, of whom 116 with paired biopsy and surgical samples were analyzed. A custom MSI-NGS panel was employed and its performance was compared to MSI polymerase chain reaction (MSI-PCR), which served as the gold standard. Results: Out of the 116 cases, 112 patients showed consistent MSI status between biopsy and surgical samples, with an overall accuracy of 97% regardless of the detection method used. In the cases with MSI discrepancy (6 cases), 83% (5/6) of the patients showed a transition from MSS to MSI-H from biopsy to surgery. Interestingly, all eight patients who received neoadjuvant chemotherapy maintained an unchanged MSI status. However, in one patient who received adjuvant treatment and underwent a repeat biopsy after surgery, the MSI status exhibited alterations. Further analysis of clonal evolution revealed that this heterogeneity stemmed from the disappearance of the original clones and the emergence of new clones. Additionally, the NGS panel exhibited strong concordance with MSI-PCR and high accuracy, sensitivity and specificity with an AUC of 0.942 after rigorous validation. Conclusions: The study revealed a high concordance in MSI status between paired biopsy and surgical samples by employing a custom MSI-NGS panel for MSI status detection, providing reliable basis for further research in this field. Moreover, neoadjuvant chemotherapy seemed to have no impact on MSI status in our study, whereas postoperative adjuvant chemotherapy may influence changes in MSI status, highlighting the necessity of reevaluating MSI status after surgery. The findings also underscore the potential of NGS-based MSI detection as a valuable tool for clinical decision-making.