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The optimal conditions for extraction, isolation and determination of chemical components of Gastrodia tuber with HPLC (High-performance liquid chromatography) were established. Using these conditions the chemical fingerprints and Gastrodin content of Gastrodia tuber samples from 6 different provinces or regions were determined. The results show that the chemical fingerprints of all tested Gastrodia tubers have five common peaks with very approximate retention time. The chemical fingerprints of Gastrodia tuber from the same province are quite similar, but those of Gastrodia tubers from the different provinces are not. Several better Gastrodia tuber populations were found with higher gastrodin content. The findings can be used to identify the quality of Gastrodia tuber, authenticate genuine Gastrodia tuber, determine the Gastrodine content and discover the better Gastrodia tuber populations. The same techniques may be applied to similar research of other medicinal plants.