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It is important to identify epidermal growth factor receptor (EGFR) mutations, which have a good value for the individualized management of patients with non–small-cell lung cancer. A novel method for detecting the mutations on exons 19, 21 of EGFR in primary carcinoma samples by a fluorescence polarization (FP) assay was developed in this research. Firstly, 2 pairs of general primers of exons 19, 21 of EGFR were, respectively, used to amplify the target regions in each exon in 2 reactions. Then, 2 probes specific for wild or mutation exons 19, 21 of EGFR were labeled with tetramethyl 6-carboxyrhodamine or 6-carboxyfluorescein hybridized, respectively, with their target amplicons, and the hybridization resulted in an increase in the FP values. Exon 19 deletion and exon 21 missense mutation were determined by the analysis of the FP values. EGFR mutations in 372 non–small-cell lung cancer samples were analyzed in parallel with an FP assay and a sequencing assay. There was no significant difference between the mutation status results obtained with the FP assay and the results obtained with the sequencing assay. The minimum detection level established with this assay was 40 copies/uL. Reliable results could be obtained when more than 30 ng of DNA was tested by a FP assay. An FP assay was able to detect the mutation DNA of EGFR even when its content was as low as 10%. An FP assay allowed the semiautomated detection of EGFR mutations in solution, and it was much simpler and cost effective than the traditional methods.