Background: Targeted therapy has improved outcomes in EGFR mutated and ALK translocated NSCLC but options for squamous cell carcinoma and KRAS-mutated tumors remain scarce. Genome re-replication may cause gene amplification, chromosomal translocation and loss, contributing to tumor progression. Cell cycle checkpoints and DNA damage response are activated by induced re-replication. CHK1/2-mediatedcell cycle checkpoints prevent re-replication and maintain genomic integrity. Specific CHK1 inhibitors delay tumor growth in combination with pemetrexed in NSCLC xenograft models. Methods: In a panel of NSCLC adenocarcinoma and squamous cell carcinoma cell lines with TP53, KRAS andEGFR mutations and six PC9-derived, TKI resistant cell lines treated with CHK1 and CHK1/2 inhibitors; IC50 was determined by MTT assay. We analyzed expression of genes involved in the re-replication pathway (MDC1, ATR, ATM, CHEK2, Rap80, Cdc1, Cdc6, MYC, SLX4, CHEK1, BRCA1, BRCA2, p53, ORC4, ORC5, ORC6, GMNN) by RT-PCR. Results: In KRAS-mutated cell lines, CHK1 inhibition resulted in very low IC50 for H23 0.2 µM, for A549 2.4 μM and for Calu6 0.3 μM; single CHK2 inhibition was less successful. Interestingly, a p53 mutated squamous cell line (SK MES1) had high basal expression levels of CHK1 and CHK2 and the lowest IC50 (0.024 μM) with CHK1 selective inhibitor. IC50 was significantly higher in SK MES1 cell line (84.62 µM vs 0.024 µM) with dual CHK1-CHK2 vs single CHK1 inhibition. Two EGFR-resistant cell lines, one with T790M mutation, were highly sensitive to CHK1inhibition (IC50 0.19µM, PC-GR5; 0.40 µM, PC9-GR4). We observed different expression levels of key genes of the re-replication pathway. Conclusions: Different expression of genes involved in the re-replication pathway and sensitivity of some squamous NSCLC cell lines to selective CHK-1 and dual CHK1-CHK2 inhibitors indicates a potential new therapeutic approach for these subgroups. Our experiments also showed that CHK1 inhibition has strong activity in KRAS mutated cell lines (H23, Calu6, A549), particularly when a P53 mutation is also present. Further studies are warranted after these findings.