Rationale: Gefitinib is effective in treating non-small cell lung cancer (NSLCL) patients with epidermal growth factor receptor (EGFR) mutations. Deletions in exon 19 and L858R in exon 21 are the most well documented EGFR mutations that are associated with good gefitinib responsiveness. Objectives: In order to clarify the influence of gefitinib timing, we conducted the study to compare outcomes of different lines of gefitinib treatment in patients with exon 19 deletions or L858R. Methods and Measurements: We surveyed the clinical data and mutational studies of NSCLC patients with EGFR mutations in the National Taiwan University Hospital. Results: 328 patients, who received gefitinib for stage IIIb or IV for NSCLC, were adequately sequenced for EGFR mutations. 192 patients had mutant-EGFR, including 77 patients with exon 19 deletions and 75 patients with L858R. The total 152 patients with exon 19 deletions or L858R receiving gefitinib were classified into chemonaive group (91 patients) or chemotherapy-treated group (61 patients). Chemonaive status before gefitinib and female gender were associated with clinical response to gefitinib (p = 0.006 and 0.053 respectively). The overall survival after the start of anti-tumor therapies or progression-free survival after gefitinib therapy were not significantly different between these two groups (p = 0.207 and 0.804 respectively). Clinical response to gefitinib was the only factor associated with better overall survival (p = 0.001). Conclusions: The study suggested that gefitinib is effective in mutant-EGFR patients. Gefitinib response rate in chemonaive patients is higher than chemotherapy-treated patients, however, there is no difference in overall survival.