The exact clinical significance of EGFR mutation status in NSCLC at the time of initial diagnosis remains disputable. The gene expression module in NSCLC for chemotherapy outcome prediction needs to be developed. We analyzed 56 patients with NSCLC received chemotherapy either with (n=20) or without EGFR-TKIs (n=36) between 2008 and 2012 in China. EGFR mutation test and gene expression profiling were performed in samples obtained before medication treatment by liquidchip platform. Significant association (P = 0.028) was seen between EGFR mutation status before first-line chemotherapy and EGFR-TKIs treatment outcomes, which even can be found from the status before second- or third-line treatment. A14-gene expression profiling had been studied. Patients with low mRNA expression of ERCC1 or TYMS preferred higher DCR to cisplatin and pemetrexed than those with high expression (P = 0.39 and P= 0.11). Highly co-expression of TUBB3 and STMN1 gene has associated with the resistance to antimicrotubule drugs (P = 0.03). Our data suggest the EGFR mutations status, even at the time of initial diagnosis, is predictive of outcomes of TKIs treatment after chemotherapy. The mRNA expression profiling investigated in this study has a predictive value in NSCLC treatment, but further research with expanded samples is still required.