Universal screening for Lynch syndrome through mismatch repair (MMR) immunohistochemistry (IHC) on tumor samples has brought to light several heterogenous MMR staining patterns. At our institution, a prospective study of universal Lynch syndrome screening using MMR IHC on 125 endometrial cancers (EC) led to the identification of subclonal loss of MMR protein expression within the tumor (n=9). We also interrogated the MMR staining patterns in MMR-deficient EC with concurrent endometrial intraepithelial neoplasia (EIN; n=14) and all mixed-type ECs (n=14) to look for concordant or discordant profiles between the various components. MLH1 promoter methylation and microsatellite instability testing was performed on discordant subclones. Abrupt and complete subclonal loss of MMR expression was identified in 9 cases (7.2%; 7 subclonal MLH1/PMS2 loss, 1 subclonal loss of MLH1 and complete loss of PMS2, and 1 subclonal MSH6 loss). All subclonal MLH1 losses were associated with epigenetic silencing. In cases with concomitant EIN (n=14), 7 cases showed concordant MMR IHC between EC and EIN, and 4 cases showed MMR protein loss confined to the EC. The remaining 3 cases demonstrated subclonal staining in the EIN. In mixed tumors (n=14), subclonal or total MMR IHC deficiency was confined to endometrioid components. In summary, discrete subclonal loss of MMR protein expression occurs in up to 7.2% of EC and, in our experience, only in endometrioid components. Importantly, subclonal MLH1 MMR defects appear to be a biological phenomenon that can be explained by methylation and somatic events, without evidence of underlying germline alterations.