An RP-HPLC method was developed for the first time to simultaneously determine five major compounds in Polygonum cuspidatum, namely resveratrol, polydatin, anthraglycoside B, emodin and physcion with UV detection at 306 nm. The column was an Agilent Zorbax SB-C18 (250 × 4.6 mm i.d., 5 μm). The separation was carried out with a gradient program. The mobile phase was acetonitrile–water (containing 0.1% formic acid) at a flow rate of 1.0 mL min−1. The standard curve was rectilinear in the range of 2.04–62.96 μg mL−1 (r = 0.9998) for resveratrol, 20.13–239.7 μg mL−1 (r = 0.9998) for polydatin, 7.19–71.92 μg mL−1 (r = 1.0000) for anthraglycoside B, 2.68–83.68 μg mL−1 (r = 0.9998) for emodin and 0.60–14.37 μg mL−1 (r = 0.9997) for physcion. The recoveries of the markers were 96.0, 106.5, 97.8, 97.9 and 98.1%, respectively. The relative standard deviation of intra-day and inter-day were less than 5.0 and 2.3%. This method was simple, accurate and reproducible. The developed method was successfully applied to analyze five compounds in P. cuspidatum of 20 commercial brands.