A high performance liquid chromatographic (HPLC) method was developed to determine the five endogenous hormones including indole-3-acetic acid (IAA), abscisic acid (ABA), gibberellic acid (GA3 ), zeatin (ZT) and salicylic acid (SA) in wheat. The separation conditions were optimized, and methanol was chosen as the extraction solvent. Then the extract was extracted by petroleum ether and ethyl acetate, and purified with the Sep-Pak C18 column. The chromatographic conditions were as follows: Eclipse XDB-C18 reversed phase column (250 mm x 4.6 mm, 5 microm), the flow rate of 1 mL/min, the injection volume of 10 microL, and the detection wavelength of 240 nm were used for the separation of SA from 14.5 min to 18 min, while the detection at 254 nm used for the separation of the others. Methanol (A) and acetic acid aqueous solution (pH 3.6) (B) were used as the mobile phases with the linear gradient set as follows: 0-7 min 20% A, 7-10 min 20% A-28% A, 10-17 min 28% A, 17-19 min 28% A-40% A, 19-35 min 40% A. The results showed that: the hormones were separated well with the recoveries of 96.9% - 98%, and the RSDs were in the range of 1.54% to 2.29%. It is a reliable method for rapid, accurate separation and determination of the endogenous hormones in wheat.