Biotechnique for programmed freezing of the spermatozoa of the edible mud crab Scylla serrata and the procedures for assessing viability of the frozen-thawed spermatozoa are described. Free spermatozoa recovered from spermathecae and those released from the spermatophores were subjected to cryoprotectant treatment at different physiological temperatures (30ยฐC, 23ยฐC, 15ยฐC), frozen to subzero temperatures in a rate-controlled programmable freezer Kryo 10/1.7 and subsequently plunged into liquid nitrogen. Post-thaw viability, after 8 hr of storage at -196ยฐC, was assessed by artificial induction of acrosome reaction using divalent cation ionophore A23187. Hypo/hyperosmotic sensitivity tests were used to assess the membrane integrity of the spermatozoa before and after cryopreservation experiments. Viability tests using dye exclusion methods were discontinued owing to variations in incorporation of different stains into the spermatozoa. The limitations of the dye exclusion method as an indicator of sperm viability are discussed. Dimethyl sulfoxide, ethylene glycol and glycerol offered cryoprotection at concentrations ranging from 5% to 15% v/v. Post-thaw survival was highest (52%) with 12.5% glycerol at a cooling rate of -5ยฐC/min.