To obtain well spread chromosomes, the cytogenetic protocol for Pangasianodon hypophthalmus and Clarias gariepinus were optimized. This includes, the colchicine concentration (0.01%, 0.025%, 0.05%)/exposure duration (1, 3, and 5 h), hypotonic solution (distilled water or 0.075M KCl solution)/exposure duration (30 min, 1, and 2 h), the time of cell suspension preparation (at hypotonic treatment or before slide preparation) and chromosome aging period (0, 3, and 7 days in Carnoy's fixative). In addition, the type (i.e., fin, gill or kidney) and the amount of tissue (10, 50, 100 or 150 mg) were also investigated. Regardless of the species, the result obtained showed that well-spread chromosomes could be obtained using the following optimized protocol: Juveniles are injected with 0.05% colchicine (at one ml kg-1) and allowed to swim for 3 h. Then, 50 mg of gill tissue is made into cell suspension in 0.075M KCl for 1 h. The cell suspension is treated in Carnoy's fixative (changed three times at 20 min interval) and then aged for 3 days. Finally, chromosome slides are made and stained with 10% Giemsa for 1 h.