Background: MSI-H tumors are associated with higher TMB. We examined the yet uncharacterized relationship between TMB and individual MMR gene alterations in MSI-H tumors. Methods: MSI-H was determined by examining altered microsatellite (MS) loci using NextGen sequencing (cutoff: > = 46) on a 592-gene panel. TMB was calculated by enumerating somatic missense mutations. MMR protein expression was evaluated by IHC. ANOVA and chi-square tests were used for comparisons. Results: A total of 1057 MSI-H tumors (283 colorectal cancer [CRC]; 449 endometrial cancer [EC]; and 325 others from 29 cancer types) were examined. High TMB (≥ 17 mutations/megabase [mt/MB]) was seen in 74% of tumors. MSI-H CRC had the highest TMB compared to MSI-H EC and “all others” (mean TMB: 39 vs. 23 vs. 31 mt/MB, respectively; p < 0.0001). There was no difference in TMB between BRAF V600 mutant and wild type MSI-H CRC (38.7 vs. 39 mt/MB). In general the most frequently altered (IHC loss or mutation) MMR genes were MLH1 and PMS2 (72% and 83%, CRC; 90% and 95%, EC). MSH2 and MSH6 were more frequently altered in CRC than EC (21% vs. 5% and 49% vs. 28%, respectively; p < 0.0001). In CRC, MSH2 and MSH6 were more frequently altered in left than right sided MSI-H tumors (45% vs. 12% and 67% vs. 40%; p = 0.01). Overall MSH2 or MSH6 alterations were associated with higher TMB (48.5 and 40 mt/MB, respectively) than MLH1 or PMS2 (27 mt/MB for both); P < 0.0001. Tumors with MSH2/6 co-alterations (4%) had a higher TMB compared to those with MLH1/PMS2 (39%) co-alteration (50 vs. 24 mt/MB; p < 0.0001). PD-L1 overexpression was seen at a higher frequency in tumors with MSH2 (23%) than MSH6 (16%), MLH1 (16%), or PMS (14%); P = 0.01. MSH2/6 alterations in EC were associated with higher MS alterations (MSH2, 88; MSH6, 73; MLH1, 68; PMS2, 68; p < 0.0001), while all genes were equal in CRC. MSH2 alterations were associated with higher frameshift mutation rates in 36 genes in EC, and in different 10 genes in CRC. Conclusions: TMB varies significantly across MSI-H tumors. MSH2/MSH6 alterations were associated with a significantly higher TMB than MLH1/PMS2 across several cancer types. The MS alterations associated with MSH2/6 were tumor-type specific.