The aim of this study was to compare the HPLC fingerprints of different Rehmannia Species to build a standard for strengthening the quality control and species identification. The method was HPLC and the Chinese Medicine Chromatography Similarity Evaluation System and similarity and clustering analyses to the HPLC fingerprint spectrum of different kinds of Rehmannia was performed. An optimized HPLC technique was obtained, and the Rehmannia HPLC comparison fingerprint spectrum containing 18 featured fingerprint peaks was established. The 9 agriculture species were first divided into two broad categories and then further divided into four detailed categories. It was concluded that the established spectrum could be used as the standard spectrum for Rehamnin quality control since the chemical fingerprint spectrums of different Rehmannia species were different, and could provide theoretical and technical support for quality control, species identification and classification of Rehmannia.