The treatment of advanced nonsmall cell lung cancer (NSCLC) with PD-1/PD-L1 immune checkpoint inhibitors has improved clinical outcome for a proportion of patients. The current challenge is to find better biomarkers than PD-L1 immunohistochemistry (IHC) that will identify patients likely to benefit from this therapy. In this exploratory study we assessed the differences in T-cell subsets and PD-1 expression levels on T-cells in tumour-draining lymph nodes (TDLNs) and peripheral blood mononuclear cells (PBMCs). To evaluate this, flow cytometric analyses were performed on endobronchial ultrasound-guided (EBUS) fine-needle aspirates (FNA) from TDLNs of patients with NSCLC, and the results were compared to paired PBMC samples. For a select number of patients, we were also able to obtain cells from a non-TDLN (NTDLN) sample. Our data show that the frequency of PD-1+ CD4+ and CD8+ T-cells, as well as the PD-1 expression level on activated regulatory T (aTreg) and CD4+ and CD8+ T-cells, are higher in TDLNs than in PBMCs and, in a small sub-analysis, NTDLNs. These elevated PD-1 expression levels in TDLNs may reflect tumour-specific T-cell priming and conditioning, and may serve as a predictive or early-response biomarker during PD-1 checkpoint blockade.