Here, we constructed the single-cell and spatially resolved transcriptome atlas of human non-small cell lung cancer (NSCLC) with well-designed multiple regional sampling, from 31 single-cell RNA sequencing samples and 18 spatial transcriptome sequencing samples. We identified malignant subclusters exhibited heterogeneity in genetics, pathological subtypes, transcriptional profiles and niche-specific features. In tumoral area, neutrophil was the dominantly infiltrated cell group while in tumor surrounding area, myeloid cell dominated. However, the superiority of neutrophil in tumor was abolished by genomic alteration events, like BRAF and EGFR mutations. Furthermore, we found that CXCL1+ cancer associated fibroblasts (CAFs) had strong interaction with its neighboring malignant subclone, hence affecting the phenotype of the latter one. The terminally exhausted CD8+ T cell accumulated in central tumor regions, exhibiting the most powerful neoantigen-reactive function.