Synthetic pyrethroid insecticides are chiral compounds with multiple asymmetric positions. In this study, isomers of four commonly used pyrethroids were separated at the enantiomeric level by enantioselective high-performance liquid chromatography (HPLC), and differences between enantiomers in aquatic toxicity were characterized using individual isomers. Isomers of cis-bifenthrin and permethrin were completely resolved on a Sumichiral OA-2500-I column. All eight isomers of cypermethrin and cyfluthrin were completely separated on two chained Chirex 00G-3019-DO columns. Great differences were found between enantiomers in the acute toxicity to aquatic invertebrates Ceriodaphnia dubia or Daphnia magna. In cis-bifenthrin (cis-BF) and cis-permethrin (cis-PM), the 1R-cis isomer was 15 -38 times more active than the 1S-cis enantiomer, while in trans-PM, the 1R-trans isomer was substantially more toxic than the 1S-trans enantiomer. In cypermethrin or cyfluthrin, two of the eight isomers, 1R-cis-aS and 1R-trans-aS, contributed for almost all the toxicity in the racemate, while the other six enantiomers were inactive. These results suggest that significant enantioselectivity occurs for pyrethroids in aquatic toxicity, and such enantiomeric differences must be considered when evaluating ecological effects of pyrethroid insecticides.