A simple PCR-based lateral flow assay (LFA) was developed to detect turkey ingredient in foods. We developed this assay based on PCR amplification of turkey DNA, followed by detecting labeled amplicon by LFA. The results could be observed by the naked eye within 5 min following PCR. The reliability of this assay was confirmed by gel electrophoresis. Specificity tests validated no cross-reactivity with 21 other animal and plant samples. As low as 0.1% (w/w) of turkey in mixed meats were detected by the proposed PCR-LFA. Heat treatment did not affect the sensitivity of this assay for detecting turkey ingredient. The reproducibility of PCR-LFA was confirmed by the fully consistent intra-laboratory tests and acceptable inter-laboratory tests. Finally, this assay was successfully used to authenticate 24 commercial turkey products. No turkey ingredient was identified in two frozen turkey rolls, indicating food fraud cases. PCR-LFA provides an accurate, sensitive and cost-effective approach for turkey authentication. It is a promising tool for species authentication, food quality assurance and traceability.