Background: The presence of the programmed death ligand-1 (PD-L1) has been used as biomarker to select patients and analyze responses to anti-PD-1/L1 antibodies. PD-L1 positivity may be a result of genetic mechanism leading to constitutive PD-L1 expression or inducible mechanism by T-cell presence. Each mechanism may have different significance and response to such therapy. In colorectal cancer (CRC), targeting immune checkpoint inhibitor has been recommended for patients with microsatellite instable (MSI). This study aimed to investigate the mechanism of PD-L1 gene activation in colorectal cancer (CRC) patients, particularly in MSI population. Methods: We have investigated 61 archived formalin fixed paraffin embedded CRC specimens of patients from Medistra Hospital, Jakarta admitted in 2010 - 2016. Immunohistochemistry was performed to measure expression of PD-L1 in tumor cells as well as MSI status using antibodies against PD-L1 and MMR (MLH1, MSH2, PMS2 and MSH6), respectively. Subset of PD-L1 positive patients was then assessed for copy number variations (CNVs) using Single Tube TaqMan CNA Gene CD247PD-L1. We also observed KRAS mutation to profile possible genetic mechanism leading to the presence or absence of PD-L1 expression. Results: Analysis of 61 CRC patients revealed 15 patients (24%) expressed PD-L1 on their tumor cell membranes. The prevalence of surface membrane PD-L1 was significantly higher in patients with MSI (87%; 7/8) compared to patients with microsatellite stable (MSS) patients (15%; 8/53) (P = 0.001). Although amplification of PD-L1 gene was not found among PD-L1 positive patients, low level amplification of PD-L1 gene was commonly observed in MSS patients (75%; 6/8) than in MSI patients (43%; 3/7). We found 26% of CRC patients harbored KRAS mutations (16/61), so far the distribution of KRAS status did not correlated with PD-L1 expression. Conclusions: Our data suggest genetic mechanism through amplification of PD-L1 seems not to be the mechanism underlying upregulation of PD-L1 expression in CRC patients. However, further studies are warranted to confirm the results.