Abstract: A method for HPLC fingerprint determination of different growing habitats on Perilla frutescens leaves was established using HPLC detection method equipped with UltimateTM XBC18 column (4.6 mm×250 mm, 5 μm). Acetonitrile and water containing 01% acetic acid were used as mobile phase, and rosmarinic acid served as the reference standard. HPLC had gradient elution mode with the detection wavelength of 330 nm and 9 samples collected from different habitats were detected. Data were analyzed by fingerprint similarity evaluation software to compare the samples. The mutual mode of HPLC fingerprints was set up and 12 mutual peaks were indicated. The P. frutescens samples had significant difference in fingerprints. HPLC detection method is accurate and simple. It can easily identify leaf samples of P. frutescens collected from various habitats, and also can provide some useful information for quality evaluation of P. frutescens.