It remains unclear how programmed death-ligand 1 (PD-L1) expression interacts with anaplastic lymphoma kinase (ALK) mutation, its variants, and the outcome of treatment. One hundred and twenty four out of 1255 patients (9.9%) were deemed ALK-positive by the Ventana IHC assay. PD-L1 status and ALK variants were available in 100 and 59 patients, respectively. PD-L1 positive (TPS ≥ 1%) and strong positive (TPS ≥ 50%) rate was 50% and 16%, respectively. A total of 64 variant types were detected in 59 patients. V1 (32.8%) and V3a/b (28.1%) were the most common variants. There was no significant association between ALK variants and the PD-L1 expression. The presence of V3a/b subtype independently predicted a worse overall survival in patients receiving ALK inhibitor(s) (aHR 5.10 95% CI 1.22-21.25, P = 0.025) and platinum plus pemetrexed (aHR 9.62 95% CI 1.90-48.80, P = 0.006). While incorporating ALK variants and PD-L1 expression together, patients with non-V3a/b/positive PD-L1 showed a trend towards longer OS. In conclusion, ALK-positive NSCLC patients possess a high PD-L1 expression rate. Although there was no significant association between PD-L1 expression and ALK variants, the outcome of ALK-positive patients could be sorted by these two biomarkers.