In the present study, two freshwater fishes Channa punctatus and Clarias batrachus were exposed to sub-acute concentrations of a commercial-grade λ-cyhalothrin, REEVA-5, for 96 h to observe the changes in amino acid catabolism under pyrethroid-induced stress and to investigate the comparative mechanisms of ammonia detoxification in both fishes. The experiments included the estimation of levels of free amino acid, urea, ammonia and the specific activities of aspartate aminotransferase (AAT), alanine aminotransferase (AlAT), glutamate dehydrogenase (GDH), glutamine synthetase (GS) and arginase in different vital organs of fishes. λ-cyhalothrin caused significant decline in the levels of amino acids along with simultaneous significant increase in the activity of AAT, AlAT and GDH, which indicated amino acid catabolism as one of the important mechanisms to meet out immediate energy demand of fishes. The level of ammonia was observed to be enhanced considerably at lower concentrations of λ-cyhalothrin while higher concentrations caused remarkable decline. The λ-cyhalothrin treatment resulted in significant increase in the activities of GDH and GS with concomitant increase in the activity of arginase and level of urea, indicating activation of two different mechanisms of ammonia detoxification. The mechanism of ammonia detoxification through its conversion into glutamate and glutamine was more prominent in C. punctatus, while C. batrachus demonstrated ureogenesis as the major route. In fishwise comparison, C. batrachus was observed to be more sensitive with respect to the above-mentioned parameters. Another important finding was that unlike the liver, the kidney appeared as one of the primary sites of ureogenesis in fishes.