Microsatellite instability (MSI) is an important biomarker in colorectal cancer (CRC), influencing prognosis and treatment decisions. While conventional MSI detection typically relies on the pentaplex panel, newer markers like HSP110 T17 (HT-17) and CAT-25 may offer simpler, more cost-effective alternatives. This study aimed to assess the effectiveness of HT-17 and CAT-25 for detecting MSI in sporadic CRC and to explore any links between MSI status and clinicopathological features. A total of 96 Tunisian sporadic CRC patients were included, with MSI status evaluated using HT-17, CAT-25, and the refined mononucleotide repeat pentaplex panel through microsatellite genotyping. Clinicopathological data, such as tumor location and age at diagnosis, were also analyzed for associations with MSI. Among the 96 patients, 9 (9.38%) showed MSI, while 87 were microsatellite stable (MSS). HT-17 demonstrated 100% accuracy and sensitivity, matching the pentaplex panel's performance, while CAT-25 showed limited detection ability. MSI status was significantly linked to tumors in the proximal colon and, unexpectedly, to younger patients (<
50 years old). HT-17 proved to be a reliable MSI marker in CRC, offering equivalent performance to the pentaplex panel, with the added advantages of simplicity and cost efficiency. The associations between MSI, tumor location, and younger age at diagnosis may provide valuable insights into CRC biology and clinical management. Further studies with larger cohorts are needed to validate HT-17' s clinical potential, with the goal of improving personalized treatment strategies and prognostic accuracy for CRC patients.