The purpose of the present study was to identify a potential biomarker that is more effective than those already available for the prognosis of non‑small cell lung cancer (NSCLC) patients. The expression of never in mitosis gene A (NIMA)‑related kinase 2 (NEK2), minichromosome maintenance complex component 7 (Mcm7) and Ki67 was evaluated in 270 NSCLC tissues using immunohistochemical and immunofluorescence techniques. Associations between protein expression and clinicopathological characters were assessed, and the impact on overall survival was analyzed. High levels of NEK2, Mcm7 and Ki67 expression were detected in 25.9, 35.2 and 24.4% of the NSCLC tissues. Overexpression of NEK2 was detected more frequently in cases with high T and N stages (P< 0.0001 and P= 0.011, respectively). Correlations were present between the expression of NEK2, Mcm7 and Ki67. Kaplan‑Meier curves indicated that the patients with overexpressed NEK2, Mcm7 and Ki67 had a poorer overall survival time compared to those with low expression for all stages (P< 0.0001). In particular, the patients with NEK2 overexpression had a poorer prognosis. Multivariate Cox regression analysis showed that NEK2, Mcm7 and Ki67 are independent prognostic indicators for NSCLC. In conclusion, the data indicate that compared with Mcm7 and Ki67, NEK2 may be a more effective tumor proliferation marker of poor prognosis for NSCLC patients, and that NEK2 may represent a novel potential target for NSCLC therapeutic intervention.