HPLC fingerprinting and quantification of gentiopicroside(GPS) and loganic acid(LA) in Gentianae Macrophyllae Radix(GMR) crude drugs were developed in this study.The samples were separated on Zorbax SB-C_(18) column(250 mm×4.6 mm, 5μm) with a linear gradient of acetonitrile and 0.04%phosphoric acid.The HPLC flow rate was 1.0 mL/min and a UV absorption was measured at 230 nm.An orthogonal L9(3^4) test was applied for the optimization of sample extraction conditions,and an aliquot of GMR sample(g) was extracted with 15-fold of 50%ethanol(mL) for 30 min by sonication.Quantitative analysis showed that the content of GPS(14.05 mg/g-74.61 mg/g) in all samples was obviously higher than that of LA(1.13 mg/g-40.46 mg/g). Based on the content ratio of GPS over LA(1.8-11.4),samples originated from Gentiana macrophylla(with content ratio of GPS over LA≤4.3) could be distinguished from those from G.dahurica and G.dahurica var.gracilipes(with content ratio of GPS over LA≥4.8).The principle components analysis of the HPLC fingerprints showed that samples originated from G.macrophylla and G.dahurica(including G.dahurica var.gracilipes) could be divided into two groups.This established HPLC-DAD method could be efficiently used for the species identification and quality control of GMR crude drugs.