Qualitative and quantitative analyses of quercetin (Qu) and isorhamnetin (Is) in Hippophae rhamnoides L. fruits hydrolysis products from 10 different origins were conducted by high-performance thin-layer chromatography (HPTLC), using 3% sodium acetate silica gel G plate (Anhui Liangchen), dichloromethane–ethyl acetate–formic acid (7:3:1, V/V) as the developing reagent, 1% ethanol solution of aluminum trichloride as the chromogenic agent, scanning at a wavelength of 390 nm. The content of Qu and Is in H. rhamnoides L. fruits hydrolysis products from 10 different producing areas was determined. The results showed that the RF value of Qu was 0.48 ± 0.05, the linear range was 0.06–0.42 μg/μL, and the correlation coefficient was 0.9990, indicating that the linear correlation of Qu was good. The RF value of Is was 0.64 ± 0.05, the linear range was 0.04–0.44 μg/μL, and the correlation coefficient was 0.9993, indicating that the linear correlation of Is was good. The precision analyses of Qu and Is showed that the RSD was < 5.0%, indicating the good precision of the instrument. The repeatability analyses of both Qu and Is showed an RSD < 3.0%, indicating that the method is reproducible. The limit of detection (LOD) of Qu was 50 ng, and the limit of quantification (LOQ) was 165 ng. The LOD of Is was 50 ng, and the LOQ was 167 ng. The stability study showed that the sample was stable within 90 min at room temperature, with an RSD < 2.0%. The average recovery of Qu was 99.99%; the average recovery of Is was 100.79%. The results showed that the Is content was higher than the Qu content in the Inner Mongolia and Shanxi origins, and the Qu content was higher than the Is content in the eight other origins.