High-performance thin-layer chromatography, which is a quick qualitative and quantitative method, was used to determine four kinds of hydroxynaphthoquinone ingredients, namely β-hydroxyisovaleryl shikonin, L-shikonin, β-acetoxyisovaleryl shikonin, and acetyl shikonin, in Arnebia euchroma (Royle) Johnst. roots originating from nine batches in Urumqi, China. The developing solvent was cyclohexane‒dichloromethane‒ethyl acetate‒formic acid (5:9:0.6:0.11, V/V), the temperature was 25 ± 1℃, the humidity was 10%, and the detection wavelength was 520 nm. The linear ranges of β-hydroxyisovaleryl shikonin, L-shikonin, β-acetoxyisovaleryl shikonin, and acetyl shikonin were 0.05‒0.6, 0.0625‒0.75, 0.125‒1.5, and 0.05‒0.6 μg, respectively, and the linear correlation coefficients were 0.9988, 0.9962, 0.9967, and 0.9982, respectively. The results showed good linear relationships. Precision analyses revealed a relative standard deviation (RSD) of < 3.00%. The sample was stable within 24 h at room temperature at RSD < 3.00%. The average recoveries of β-hydroxyisovaleryl shikonin, L-shikonin, β-acetoxyisovaleryl shikonin, and acetyl shikonin were 99.53%, 100.57%, 98.59%, and 96.97%. This method is reliable and can be used to determine four hydroxynaphthoquinone ingredients in the roots of A. euchroma (Royle) Johnst. from different batches.