In order to preserve the genetic diversity of cichlid fish in gene banks, it is necessary to use certain extenders to maintain the integrity of spermatozoa cells during cooling.To evaluate the effects of different extenders on the quality parameters of cooled semen of Geophagus brasiliensis.Semen samples were collected from seven adult fish and diluted with five extenders: Beltsville Thawing Solution (BTS™), Hanks' Balanced Salt Solution (HBSS), Tris-glucose, Ginsburg's Fish Ringers, and Phosphate buffered Saline. All parameters were evaluated in fresh semen samples and after cooling at 4°C at 0, 24, 48, and 96 h to evaluate cell viability (membrane integrity, DNA, and mitochondrial functionality) and motility rate and weather motility.The BTS and Tris-glucose resulted in the best outcomes (P<0.05) in terms of membrane integrity assessments (35.1% and 30.9%, respectively), DNA integrity (71.6%; 75.7%), mitochondrial function (26.9%; 28.0%) and motility rate (8.6%; 8.6%), respectively, for semen cooled to 4°C for 96 h. However, the 48-h period motility after cooling in BTS was superior to all other treatments.BTS and Tris-glucose can be considered as the best extenders for the cold storage of Geophagus brasiliensis spermatozoa.