Twelve of the fourteen stereoisomers of all-trans-, 9-cis-, 13-cis- and 15-cis-astaxanthin were simultaneously resolved on a (D)-phenylglycine covalently bonded chiral stationary phase using a ternary solvent system. The separation was optimized by systematic variation of the mobile phase modifiers. The optimized method was used to measure ratios of stereoisomers for each of the resolved mono-cis and all-trans forms of synthetic astaxanthin extracted from Carophyl Pink, a stabilized color additive used in salmon feed. In previous methods, the stereoisomeric ratios of the all-trans-astaxanthin peaks were exclusively used to distinguish between wild and aquacultured salmon. However, the stereoisomers of 9-cis-astaxanthin are sufficiently resolved on Sumichiral OA-2000 column packed with (D)-phenylglycine to permit calculation of the stereoisomeric ratios. Therefore, the stereoisomers of the 9-cis-astaxanthin may also be used to distinguish wild salmon from aquacultured salmon fed synthetic astaxanthin on Sumichiral OA-2000 columns.