Non-small cell lung cancer (NSCLC) is a potentially fatal disease and the incidence is increasing annually. In order to diagnose and treat NSCLC effectively, greater understanding of its molecular mechanism is required. In the present study, 36 NSCLC tissues and 10 normal tissues were selected. Reverse transcription‑quantitative polymerase chain reaction (RT‑qPCR) was used to analyze the CD44 mRNA expression level in NSCLC tissue and DNA sequencing was performed to further verify the CD44 expression level. Differentially expressed genes between tumor tissues and controls were determined by DNA sequencing and the Gene_act_net between CD44 and its associated genes was constructed. Gene Ontology (GO) term enrichment analysis of the differentially expressed genes was performed by the Biological Networks Gene Ontology tool. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis was performed based on the Expression Analysis Systematic Explorer test applied in the Database for Annotation, Visualization and Integrated Discovery. RT‑qPCR results showed that CD34 was overexpressed in 21 of the 36 NSCLC tissues (58.3%). The Gene_act_net indicated that there were 20 differentially expressed genes with 17 upregulated and 3 downregulated. Among them, CD44, MET, ERBB2, EGFR, AKT1, IQGAP1 and STAT3 were associated with the occurrence and migration of NSCLC. In KEGG pathway analysis, extracellular matrix‑receptor interaction and hematopoietic cell lineage pathways were the most affected by overexpressed CD44; and thus may be important in the development and migration of NSCLC. In conclusion, CD44 was overexpressed in NSCLC and the overexpression was associated with the occurrence of NSCLC and migration of NSCLC cells.