Alpha B-crystallin (CRYAB) is one of the principal members of the small heat-shock protein family, and several studies described the CRYAB expression in human cancers. However, the association between CRYAB expression and the clinical features of non-small cell lung cancer (NSCLC) is rarely elucidated. In this present study, one-step quantitative reverse transcription-polymerase chain reaction with 12 fresh-frozen NSCLC samples and Western blotting as well as immunohistochemistry (IHC) analyses in 101 NSCLC cases were conducted to investigate the relationship between CRYAB expression and the clinicopathological attributes of NSCLC. The results showed that CRYAB mRNA and protein expression levels were significantly higher in NSCLC than in matched non-cancerous tissues (p < 0.05). The IHC data indicated that the CRYAB protein expression in NSCLC was significantly correlated with TNM stage (p = 0.043), and overall survival (p = 0.029). Kaplan-Meier method and Cox multifactor analysis suggested that higher CRYAB protein level (p = 0.032) and TNM stage (p = 0.048) were statistically associated with the poor survival of patients with NSCLC. The data suggested that CRYAB may be identified as a novel prognostic marker and targeting CRYAB may provide a promising strategy for NSCLC treatment.