Hyperthermia has recently been applied to treat human non-small cell lung cancer (NSCLC). However, the mechanisms underlying cytotoxic sensitivity of NSCLC cells to hyperthermia are not fully understood. In this study, five NSCLC cell lines with different epidermal growth factor receptor (EGFR), Kirsten rat sarcoma and tumor protein p53 mutation profiles (A549, H292, H1299, PC9 and H1975) were used to evaluate effects of hyperthermia. All tested cell lines except H1975 were sensitive to hyperthermia-induced cytotoxicity. Annexin V-propidium iodide double staining, Poly(ADP-ribose) polymerase (PARP) cleavage and scanning electron microscopy revealed that apoptosis and necrosis were induced by hyperthermia in different lines. Tetramethylrhodamine, ethyl ester analysis further revealed that hyperthermia affected mitochondrial function in the four hyperthermia-sensitive lines. Transmission electron microscopic analysis revealed degeneration of cristae and ruptured mitochondria upon exposure to hyperthermia. Hyperthermia also caused elevation of reactive oxygen species in sensitive cells. In addition to these effects, hyperthermia impacted cell survival-related signalling proteins (EGFR, FAK and Akt). In particular, hyperthermia increased phosphorylated EGFR but suppressed total EGFR, phosphorylated Akt and total Akt in sensitive cells. Moreover, hyperthermia could modulate immunomodulatory molecules. Major histocompatibility complex-I (MHC-I) and surface programmed death ligand-1 (PD-L1) were both elevated by hyperthermia in all tested NSCLC cell lines except PC9. Taken together, our findings provide insights into the potential influence of different somatic mutations in NSCLC cells on hyperthermia-induced cytotoxicity and regulation of key immunomodulatory molecules.