The acquired resistance of the first generation epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKIs) is a main factor leading to poor prognosis of non-small cell lung cancer (NSCLC), so we researched whether the high expression of hypoxia-inducible factor-1α (HIF-1α) in EGFR-TKIs sensitive NSCLC tissue tends to induce the acquired resistance. We detected the HIF-1α in normal lung tissue, EGFR-TKIs sensitive NSCLC tissue, the first generation EGFR-TKIs acquired resistant NSCLC tissue and acquired EGFR T790M mutation NSCLC tissue with the method of immunohistochemistry. Then, we compared the expression of HIF-1α in these tissues, and evaluate the effect of HIF-1α expression to the occurrence of acquired resistance. The expression of HIF-1α was much higher in the EGFR-TKIs sensitive NSCLC tissue than that in normal lung tissue. HIF-1α level became higher after the occurrence acquired resistance. There was negative correlation between HIF-1α level before receiving treatment and the time of acquired resistance occurring as well as the acquired EGFR T790M mutation occurring. As the treatment going on, EGFR-TKIs sensitivity rate of low HIF-1α level group was much higher than that of high level group. The high expression of HIF-1α related with the acquired resistance of the first generation EGFR-TKIs, and HIF-1α can be a biomarker to predict the early occurrence of acquired resistance.