Some patients happen to have a colorectal cancer with microsatellite instability (MSI), but without any alteration in Mismatch Repair (MMR) system (germline mutation/promoter methylation). We aimed to identify the mechanism of inactivation of MMR genes in those cases. We studied 18 patients with MSI CCR and loss of expression of a MMR protein. DNA was extracted from tumoral and normal colonic material. We studied the 3 main MMR genes in tumors, by sequencing and large rearrangement analysis, and looked for mosaicism. Seven patients lost expression of MLH1, we found 1 mutation in the tumor for 3 patients and 2 mutations in one. Eight patients lost expression of MSH2: we found 1 mutation in 2 patients and 2 mutations in four. In the 5 cases with 2 hits, MSI was due to double somatic hits (n = 3), mosaicism (n = 1) and missed germline mutation (n = 1). Mosaicism was confirmed by HRM analysis, and by finding a germline mutation in one patient's son. We could explain MSI in the tumors of 5 patients (27.8 %). Their follow up and family's surveillance could be adjusted, as the sporadic cases don't require intensive surveillance. We describe the first case of somatic mosaicism after de novo mutation in MSH2.