MMR gene germline mutations are considered a major genetic disorder in patients with hereditary nonpolyposis colon cancer (HNPCC) or Lynch syndrome; A total of 15% of sporadic colon carcinomas are MSI-High. MSI has also been observed in other cancers, such as endometrial, gastric, and ovarian cancer. The aim of the current study was to correlate and outline the optimal method between the molecular testing of the instability of microsatellite DNA regions (MSI status) and the loss of protein expression by immunehistochemistry (MMR). A total of 242 paraffin-embedded tissues from gastrointestinal, gynecological, genitourinary, lung, breast, and unknown primary cancer patients were analyzed for the expression of MLH1/MSH2/MSH6/PMS2 by immunohistochemistry, as well as for the molecular analysis of MSI status using PCR-based molecular fragment analysis. A total of 29 MSI-High patients were detected molecularly, while 23 patients were detected by immunohistochemistry, with rates that are comparable according to the literature. Based on the agreement coefficient of the two methods, a substantial agreement emerged (Kappa = 0.675 with standard error = 0.081, p < 0.001). Despite the substantial agreement, both methods ought to be established to determine MSI-H/dMMR status in all cancer types as a first-line screening test.