Background: DNA damage and subsequent neoantigen formation has been hypothesized as a mechanismfor radiotherapy and PD-1/PD-L1 pathway inhibition to synergize in an antitumor immune response. We investigated neoadjuvant chemoradiotherapy (nCRT)-induced changes in CD8+ tumor infiltrating lymphocyte, PD-L1 and mucin expression in rectal cancer patients as compared to patients who did not receive nCRT. Methods: Tumor samples were collected from rectal adenocarcinoma patients who had undergone resection between 2008-2014 with (n = 62) or without (n = 17) nCRT treatment. Tissue sections were stained with CD8 and PD-L1 antibodies for immunohistochemistry. Whole slides images were acquired at 20x magnification. The prevalence of positive CD8 stained cells was recorded in tumor, interface tumor side, interface background rectal side. Image analysis (HALO Indica Labs) was used to determine the density (# of cells/surface area analyzed) of CD8 expressing lymphocytes. The percentage of PD-L1 membranous expression was manually counted in tumor cells (TC), tumor stroma (TS) and invasive front (IF). Mucin expression was determined as the percentage of the mucin area in the whole tumor mass area. Results: PD-L1 expression on TCs was identified in 7.7 % (6/78) of specimens. All 6 cases had received nCRT (p = 0.33). 80% and 75.5% of the nCRT cases showed PD-L1 expression on TS and IF respectively, versus 20% (p = 0.55) and 24.5% (p = 0.56) in non-nCRT cases. The median densities of CD8+ infiltrating T lymphocytes in tumor, interface tumor side, interface background rectal side did not differ significantly between the two groups (p = 0.79, p = 0.47, p = 0.22). No nCRT-changes in mucin expression observed in the 28 evaluable cases (p = 0.25). Conclusions: nCRT exposure was associated with a non-significant difference in PD-L1 expression on TS and IF cells in patients with rectal adenocarcinoma as compared to non-nCRT case, possibly due to sample size limitations. Further mechanistic investigations and comprehensive analysis of other immune checkpoints are needed to understand nCRT-induced immunologic shift in rectal cancer and to expand the potential applicability of checkpoint inhibitors in this setting.