Impacts of genetic and non-genetic intra-tumor heterogeneity (ITH) on tumor phenotypes and evolvability remain debated. We analyze ITH in lung squamous cell carcinoma at the levels of genome, transcriptome, and tumor-immune interactions and histopathological characteristics by multi-region bulk and single-cell sequencing. Genomic heterogeneity alone is a weak indicator of intra-tumor non-genetic heterogeneity at immune and transcriptomic levels that impact multiple cancer-related pathways, including those related to proliferation and inflammation, which in turn contribute to intra-tumor regional differences in histopathology and subtype classification. Tumor subclones have substantial differences in proliferation score, suggestive of non-neutral clonal dynamics. Proliferation and other cancer-related pathways also show intra-tumor regional differences, sometimes even within the same subclones. Neo-epitope burden negatively correlates with immune infiltration, indicating immune-mediated purifying selection on somatic mutations. Taken together, our observations suggest that non-genetic heterogeneity is a major determinant of heterogeneity in histopathological characteristics and impacts evolutionary dynamics in lung cancer.