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Platin-based chemotherapy is the standard treatment for patients with non-small cell lung cancer (NSCLC). However, resistance to this therapy is a major obstacle in successful treatment. In this study, we aimed to investigate the impact of several pharmacogenetic variants in patients with unresectable NSCLC treated with platin-based chemotherapy. Our results showed that <i>DPYD</i> variant carriers had significantly shorter progression-free survival and overall survival compared to <i>DPYD</i> wild-type patients, whereas DPD deficiency was not associated with a higher incidence of high-grade toxicity. For the first time, our study provides evidence that <i>DPYD</i> gene variants are associated with resistance to platin-based chemotherapy in NSCLC patients. Although further studies are needed to confirm these findings and explore the underlying mechanisms of this association, our results suggest that genetic testing of <i>DPYD</i> variants may be useful for identifying patients at a higher risk of platin-based chemotherapy resistance and might be helpful in guiding future personalized treatment strategies in NSCLC patients.