A method for determination of 5-hydroxymethylfurfural by high-performance liquid chromatography in Zhenjiang vinegar was developed. Samples were analyzed on a Venusil XBP-C18 column with oven temperature of 30℃, using a mixture of methanol and water (5∶95, v/v) as the mobile phase with the flow rate of 1.0mL/min, and detected at 284nm. Within the linear range of 0.0143～0.0858μg, the correlation coefficient was 0.9999. The method was accurate with the average recoveries rate and relative standard deviation of 98.66% and 5.53%, respectively.