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Differences in gene expression levels between the primary tumors (PTs) and matched regional lymph nodal metastases (LNs) in patients with totally excised non-small cell lung cancer (NSCLC) were explored. Microdissected formalin-fixed paraffin-embedded (FFPE) samples from (PT) and their matched infiltrated LNs, from 239 patients [183 (with matched PT and LNs samples)-case and 56 PT only samples-control cohorts] were analyzed for BRCA1, ERCC1, RAP80, PKM2, RRM1, RRM2, TS, TSP1, and TXR1mRNA expression by quantitative real-time polymerase-chain reaction (PCR). Moderately positive correlation between the expression of each gene in the PT and the matched LNs was observed. Concordance rates between the PT and the LNs were: BRCA1(67.7%), ERCC1(68.4%), PKM2(63.4%), RAP80(68.8%), RRM1(70.9%), RRM2(69%), TS(72.9%), TSP1(69.8%), TXR1(63.7%). Expression levels and their differences were correlated with Relapse-Free Survival (RFS) and Overall Survival (OS). High BRCA1PT in patients with squamous histology was associated with increased OS (p= 0.036). High TSP1PT levels were shown to be the only independent prognostic factor for OS and RFS (p= 0.023 and p= 0.007). PKM2low levels in both PT and matched LNs were associated with better OS irrespective of the underlying histology (p= 0.031). RRM1discordant levels between PT and matched LNs were associated with worse OS in squamous tumors (p= 0.019) compared to patients with both low expression in PT and LN.TXR1high levels in both PT and matched LNs were associated with better OS in patients with squamous tumors (p= 0.007).These findings indicate that there is different gene expression between PT and matched LNs which may affect the outcome in early NSCLC and therefore PT’s molecular biology should not be the sole determinant for prognostication.