Abstract: Isozymes of liver esterase from five species of freshwater fish namely, topmouth gudgeon ( Pseudorasbora parva ), goldfish ( Carassius auratus ), nile tilapia ( Tilapja nilotica ), mosquitofish ( Gambusia affinis ), and rainbow trout ( Salmo gairdneri ) were separated by discontinuous polyacrylamide gel electrophoresis. Their activities were tested using α naphthyl acetate as substrate. Two bands were separated from the liver esterase of topmouth gudgeon, goldfish, and nile tilapia, and one from the mosquitofish and rainbow trout. In vitro treatment of the isozymes with triphenol phosphate (TPP, a specific inhibitor of carboxylesterase) and malaoxon (an active metabolite of malathion) indicated the existence of a TPP sensitive esterase in each of the five species and a malaoxon sensitive esterase, which was different from the TPP sensitive one, in the topmouth gudgeon, goldfish, and nile tilapia. Significance of the distribution of the isozymes, with special emphasis on the species related difference in susceptibility to malathion was discussed.