Background: Interactions between tumor and immune cells in the tumor microenvironment (TME) play a key role in tumor progression and treatment response with accumulating evidence indicating a crucial role for tumor infiltrating immune cells. Although infiltrating cytotoxic T lymphocytes (CTLs) have been correlated with improved clinical outcome, they are ineffective in eradicating tumors due to their inhibition by immune checkpoint molecules. Immune checkpoint inhibitors have demonstrated therapeutic efficacy and durable response for several tumor types including non-small cell lung cancer (NSCLC). However, the majority of patients are resistant or relapse after initial response. Characterizing the TME for checkpoint expression with single-cell and spatial resolution can provide critical insight into new immunotherapeutic strategies and identify new predictive biomarkers for stratifying and identifying patients most likely to benefit from immunotherapy including PD-1/PD-L1 immune checkpoint blockade. Methods: Using RNAscope in situ hybridization, we evaluated in situ co-expression profiles of therapeutic checkpoint targets at single-cell level in the TME of 56 archived NSCLC FFPE tissues. Results: Checkpoint molecules including PD1, PD-L1, PD-L2, TIM3, LAG3, CTLA-4 and GITR were visualized in a highly specific and sensitive manner in individual cells within tissue morphological context. Multiple checkpoint molecules were detected in the same immune environment, especially in highly inflamed tumors. In addition to PD-L1, tumor cell-intrinsic expression of PD1, TIM3, LAG3, and PD-L2 was observed in a subset of samples. Furthermore, co-expression of therapeutic checkpoint targets including PD1, LAG3, and TIM3 was observed in infiltrating immune cells and tumor cells. Conclusions: Single-cell co-expression profiles of checkpoint molecules could shed light on how cancer cells evade the host immune surveillance and develop resistance against checkpoint blockades. Also, they could reveal valuable insights into combinatorial therapies for checkpoint markers co-expressed by the patient’s immune cells in the TME.