Gentianae Macrophyllae Radix (秦艽) is the dried root of Gentiana macrophylla Pall., G. straminea Maxim., G. crassicaulis Duthie ex Burk. or G. dahurica Fisch. It is difficult to differentiate these raw materials by morphological examination and micrography. In this study, we applied a Nested PCR and DNA sequencing method to discriminate among these raw materials of Gentianae Macrophyllae Radix and to identify these herbs in Chinese medicine preparations. The internal transcribed spacer (ITS) sequences of 18S-26S ribosomal DNA from the authentic Gentianae Macrophyllae Radix were used as standards. Samples were purchased from Chinese medicine pharmaceutical factories and local traditional Chinese herbal pharmacies. Samples of raw materials were previously identified by morphological examination and micrography. The total DNA were extracted by organic solutions and purified using a commercial kit. The ITS sequence data deduced from Nested PCR following by DNA sequencing were compared with standards and GenBank database for further identification of the origin of Gentianae Macrophyllae Radix in samples. The results obtained from morphological examination and micrography were different those from Nested PCR-DNA sequencing method in six samples of raw materials. The origin of Gentianae Macrophyllae Radix were identified as G. dahurica component present in seven samples, G. crassicaulis in two, G. straminea in two and G. triflora in one. Our methods can be applied both for the identification of raw materials of Gentianae Macrophyllae Radix and its presence in Chinese medicine preparations.