Background: LS, caused by germline pathogenic variants in DNA mismatch repair genes (MLH1, MSH2, MSH6, PMS2, EPCAM), is characterized by near universal microsatellite instability (MSI) /mismatch repair deficiency (dMMR) of associated tumors. Accurate MSI/dMMR assessment is critical to determine immune checkpoint blockade (ICB) eligibility but discordant MSI and immunohistochemical analysis of MMR status (IHC) is described. We sought to characterize MSS tumors in LS pts. Methods: Cancer-affected LS pts consented to tumor and matched normal DNA sequencing via MSK-IMPACT (NCT01775072), from 01/2014 to 10/2022 were identified with MSI status inferred via MSIsensor. Microsatellite stable (MSS) or indeterminate (MSI-I) tumors were further assessed using MiMSI, a multiple instance learning tool demonstrating high sensitivity for MSI even with low sample tumor purity. MSIsensor and MiMSI status were correlated with MMR IHC. Results: Of 405 LS cancer-affected pts, 120 had MSS, 59 MSI-I and 226 MSI-H tumors using MSIsensor. Tumor MSI status varied by LS gene with MSH6 (MSS: 33 % (40/120), MSI-I: 42 % (25/59), MSI-H: 14%, (33/226)) and PMS2 (MSS: 42% (50/120), MSI-I: 12% (7/59), MSI-H: 12%, (28/226)) more frequently associated with MSS/MSI-I than MSI-H tumors (p = 0.005) Utilizing orthogonal analysis 43% (63/147) of MSS/MSI-I tumors were re-classified as either MSI or dMMR. MiMSI was available on 106/147 MSS/MSI-I tumors resulting in the re-classification of 36 (28/38 MSI-I, 8/68 MSS tumors) as MSI. IHC was concordant with reclassified MSI status in 23/36 tumors. 5/36 reclassified cases (MSI-I on MSIsensor) had pMMR on IHC, in 2 of these cases MSI was driven by POLE/POLD1 somatic mutations. IHC was unavailable for 8/36 tumors reclassified by MiMSI. 18/36 (50%) tumors reclassified by MiMSI had low tumor content in profiled samples a known limitation of MSIsensor. Tumors reclassified as MSI on MiMSI included those considered part of the LS cancer spectrum (8 CRC, 3 gastroesophageal, 8 endometrial, 3 ovarian, 3 urothelial, 2 brain) in addition to cancers not clearly LS associated. 6 tumors classified as MSS/MSI-I on MSIsensor were MSI-I on MiMSI, 2/6 were dMMR on IHC. Amongst the 41 MSS/MSI-I tumors on MSIsensor for which only orthogonal IHC was available, 25 (11 MSS, 14 MSI-I) demonstrated protein loss consistent with the implicated LS gene. Low tumor content was noted in 44% (11/25) of tumors reclassified by IHC. Of 34 tumors found to be both MSS and MMR-proficient on orthogonal analysis, the majority (88%) were in PMS2 or MSH6 carriers, representing 9 tumor types of which 4 are considered part of the LS cancer spectrum. Conclusions: MSS tumors occur in LS pts, especially in lower-penetrance MSH6 and PMS2 carriers. Given increasing ICB utilization for MSI/dMMR tumors, orthogonal testing methods are critical to comprehensively identify all LS pts who may derive benefit from ICB particularly when sample tumor purity is low.