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Direct and label‐free detection of unmodified DNA has always been a great challenge for DNA analyses. Surface‐enhanced Raman spectroscopy (SERS) can provide structural fingerprinting of analytes with single‐molecules sensitivity, so it could be the best candidate for label‐free detection of DNA. Wild mushrooms, a very nutritious and delicious food in our lives, are widely distributed in Yunnan province. Herein, a simple and reliable method, based on SERS, was developed for label‐free detection of DNA from six wild mushrooms and three common edible mushrooms. Concentrated Ag colloid prepared by microwave heating method with high SERS activity was used to obtain high reproducible SERS spectra. SERS signatures and spectroscopic band assignments of these mushrooms DNA were analyzed. Principal component analysis was used to distinguish these nine mushrooms. This experiment provides a simple and reliable method to detect mushrooms DNA, which is useful for the study of mushrooms DNA. Also, this approach has great potential for species identification of wild mushrooms and provides a basis for learning the interaction between DNA and small molecules. To our best knowledge, it is the first SERS study on DNA from wild mushrooms. [ABSTRACT FROM AUTHOR]