Epidermal growth factor receptor (EGFR) mutations, especially deletional mutations in exon 19 (DEL) and L858R, predict gefitinib sensitivity in patients with non-small cell lung cancer (NSCLC). In this study, we validated EGFR mutation detection using high-resolution melting analysis (HRMA) and evaluated the associations between EGFR mutations and clinical outcomes in advanced NSCLC patients treated with gefitinib on a larger scale. The presence of DEL or L858R was evaluated using HRMA and paraffin-embedded tissues and/or cytologic slides from 212 patients. In 66 patients, the results were compared with direct sequencing data. HRMA using formalin-fixed tissues had a 92% sensitivity and a 100% specificity. The analysis was successfully completed in 207 patients, and DEL or L858R mutations were detected in 85 (41%) patients. The response rate (78% versus 8%), time-to-progression (median, 9.2 versus 1.6 months), and overall survival (median, 21.7 versus 8.7 months) were significantly better in patients with EGFR mutations (P < 0.001). Even among the 34 patients with stable diseases, the time-to-progression was significantly longer in patients with EGFR mutations. Patients with DEL (n = 49) tended to have better outcomes than those with L858R (n = 36); the response rates were 86% and 67%, respectively (P = 0.037), and the median time-to-progression was 10.5 and 7.4 months, respectively (P = 0.11). HRMA is a precise method for detecting DEL and L858R mutations and is useful for predicting clinical outcomes in patients with advanced NSCLC treated with gefitinib.