Microsatellite instability (MSI) is present in 15–20% of primary colorectal cancers. MSI status is assessed to detect Lynchsyndrome, guide adjuvant chemotherapy, determine prognosis, and use as a companion test for checkpoint blockade inhibitors.Traditionally, MSI status is determined by immunohistochemistry or molecular methods. The Idylla™MSI Assay is a fullyautomated molecular method (including automated result interpretation), using seven novel MSI biomarkers (ACVR2A,BTBD7,DIDO1,MRE11,RYR3,SEC31A,SULF2) and not requiring matched normal tissue. In this real-world global study, 44 clinicalcenters performed Idylla™testing on a total of 1301 archived colorectal cancer formalin-fixed, paraffin-embedded (FFPE) tissuesections and compared Idylla™results against available results from routine diagnostic testing in those sites. MSI mutationsdetected with the Idylla™MSI Assay were equally distributed over the seven biomarkers, and 84.48% of the MSI-high sampleshad≥5 mutated biomarkers, while 98.25% of the microsatellite-stable samples had zero mutated biomarkers. The concordancelevel between the Idylla™MSI Assay and immunohistochemistry was 96.39% (988/1025); 17/37 discordant samples were foundto be concordant when a third method was used. Compared with routine molecular methods, the concordance level was 98.01%(789/805); third-method analysis found concordance for 8/16 discordant samples. The failure rate of the Idylla™MSI Assay(0.23%; 3/1301) was lower than that of referenced immunohistochemistry (4.37%; 47/1075) or molecular assays (0.86%; 7/812).In conclusion, lower failure rates and high concordance levels were found between the Idylla™MSI Assay and routine tests.