Background This study examined the expression of CDC20 in human non‐small cell lung cancer (NSCLC), explored its clinicopathological significance, and evaluated as a potential prognostic marker. Methods A total of 362 cases of NSCLCs were analyzed immunohistochemically on tissue microarrays (TMAs). Additionally, the immunoreactivity of mitotic arrest defective protein 2 (MAD2) was also studied. The clinicopathological implications of these molecules were analyzed statistically. Results High‐level CDC20 protein expression (CDC20‐H) was detected in 71 cases (19.6%). Additionally, CDC20‐H was correlated with male sex, pT status, pleural invasion, and non‐adenocarcinoma (non‐ADC) histology. Significant correlation between CDC20 and MAD2 expression was found. NSCLC patients with tumor exhibiting CDC20‐H showed significantly shorter 5‐year overall survival (P = 0.0007). According to subset analyses, CDC20‐H was associated with shorter survival than CDC20‐L only among ADC patients (P = 0.0008), and not among squamous cell carcinoma (SCC) patients (P = 0.5100). Importantly, CDC20‐H was also identified as an independent prognostic factor in multivariate analysis (P = 0.0065). Conclusions CDC20 was a negative prognostic marker with significance in patients with resected NSCLC, particularly those with ADC histology. These results provide additional information for determining postoperative adjuvant treatment. J. Surg. Oncol. 2012; 106:423–430. © 2012 Wiley Periodicals, Inc.