Objective To discuss the effects of genomic instability and MYC gene mutation of non-small cell lung cancer （NSCLC） A549 cells on the resistance of gemcitabine， and to clarify the mechanism. Methods The A549 cells were continuously treated with 2 mg·L^－1 of gemcitabine （A549 group） to establish the resistant cell line A549R （A549R group）， and si-NC and si-MYC were transfected into the A549R cells to regarded as si-NC A549R group and si-MYC A549R group， respectively. CCK-8 assay was used to detect the inhibitory rates of the cells in various groups after treated with various concentrations of gemcitabine （0， 1， 2， 4， 8， 16， and 32 mg·L^－1）；flow cytometry was used to detect the apoptotic rates of the cells in various groups； transcriptome sequencing technology （RNA-seq） and Kyoto Encyclopedia of Genes and Genomes （KEGG） signaling pathway enrichment analysis were used to identify the differentially expressed genes in the A549 and A549R cells； real-time fluorescence quantitative PCR （RT-qPCR） method was used to detect the expression levels of muts homology 2 （MSH2）， muts homology 6 （MSH6）， and recombinant DNA repair protein RAD50（RAD50） in the A549 and A549R cells； Western blotting method was used to detect the expression levels of genome instability-related proteins MYC proto-oncogene （MYC） and phosphorylated H2AX （γH2AX） in the cells in various groups； chromatin immunoprecipitation （ChIP） was used to detect the enrichment of RNA pol Ⅱ and γH2AX on the MYC gene； PCR method was used to amplify and detect the mutations in MYC gene in the A549R cells. Results Compared with A549 group， the inhibitory rates of the A549R cells in A549R group treated with 2， 4， 8， 16， and 32 mg·L^－1 gemcitabine were decreased， and the apoptotic rate of the cells after treated with 8 mg·L^－1 gemcitabine was decreased （P<0.05）. Compared with A549 group， a total of 234 mRNAs in the cells in A549R group were upregulated and 205 mRNAs in the cells were downregulated， and the expression levels of mismatch repair-related genes （MSH2 and MSH6）， RAD50， and MYC were significantly increased （P<0.05）. The KEGG signaling pathway enrichment analysis showed that the upregulated genes were mainly involved in non-homologous end-joining， mRNA surveillance pathway， and DNA replication signaling pathways. Compared with A549 group， the expression levels of MYC and γH2AX proteins in the cells in A549R group were increased （P<0.05）.The ChIP assay results showed that the enrichment of RNA pol Ⅱ and γH2AX at the MYC transcription start site and exon 2， with a G254A mutation identified in MYC exon 2 was increased. Compared with si-NC A549R group， the expression levels of MYC mRNA and protein in the cells in si-MYC A549R group were decreased （P<0.05）， the inhibitory rates of the cells after treated with 2， 4， 8， 16， and 32 mg·L^－1 gemcitabine were increased（P<0.05）， and the apoptotic rates were increased （P<0.05）. Conclusion The genome instability of the A549R NSCLC cells resistant to gemcitabine is increased， and the mutations and amplification occur in the MYC gene. Knockdown of MYC can restore the sensitivity of A549R cells to gemcitabine.