Epidermal growth factor receptor (EGFR) mutations are potential markers driving carcinogenesis, and may alter the response to EGFR tyrosine kinase inhibitors in patients with non-small cell lung cancer (NSCLC). The frequency of EGFR mutations in patients with NSCLC differs according to sex, smoking habits and regional-based ethnicity differences. The aim of the present study was to determine the frequency of EGFR mutations in Turkish patients with NSCLC to highlight the importance of regional differences, and their associations with patient characteristics. Genomic DNA was extracted from formalin-fixed and paraffin-embedded tumor tissue sections of 409 NSCLC patients. The most common EGFR mutations in exons 18, 19, 20 and 21 were detected using BioFilmChip-based microarray assay. The overall EGFR mutation frequency was 16.6%, and the highest mutation frequencies were observed in exon 19 (6.4%) and exon 21 (7.3%). There was a higher frequency of EGFR mutations in females compared with males and in never-smokers compared with smokers (both P <= 0.05). These results were similar to other European population-based studies, but not consistent Middle-Eastern based studies. The present study may contribute to understanding the gradient frequency of EGFR mutation across different ethnicities, and in designing genome wide-based collaborations that may reveal novel decision making and susceptibility mutations in EGFR in patients with NSCLC.