A silver ion HPLC procedure is described that is suitable to determine the fatty acid composition of plant seed oils. After conversion of fatty acids to p-methoxyphenacyl derivatives, it was possible to achieve baseline resolution of all fatty acid components with 0 to 3 double bonds, including the positionally isomeric 18:1 fatty acids oleic acid (cis 9-18:1), petroselinic acid (cis 6-18:1), and cis-vaccenic acid (cis 11-18:1), in aniseed oil (Pimpinella anisum, Apiaceae) by a single gradient run on a single cation exchange column laboratory converted to the silver ion form. The UV detector response (280 nm) was linearly related to the fatty acid concentration in the range 0.01 to 3.5 mg/mL.