The use of technologies in assisted reproduction, such as the use of centrifugation, has been reported as a tool that allows separating the best portion of sperm from cryopreserved milt. The main of this work was to compare two separation techniques, differential and density gradient, on thawed Rhamdia quelen milt, based on sperm functionality and fertilization capacity. Three treatments were compared: control, differential centrifugation or pelletization (T1), and density gradient centrifugation (T2). Motility percentage was better for differential (42.58 +/- 6.50%) than for the control treatment (32.44 +/- 6.03%). Membrane integrity and kinetic parameters was not different between treatments. The presence of normal sperm was higher in T2 treatment (13.55 +/- 5.82%) when compared to the control (10.72 +/- 3.53%) and T1 (3.97 +/-& nbsp;1.95%). There was a lower rate of morphological tail damage in the T1 (26.91 +/- 7.13%) treatment compared to the T2 (37.48 +/- 10.81%) and control (45.88 & PLUSMN; 4.84%). For the head morphologies, the lowest percentages were for the control (35.72 +/- 5.89%) and T2 (4.89 +/- 14.94%) treatments compared to the T1 treatment (62.59 +/- 9.67%). In the MTT (3-(4, 5-dimethylthiazolyl-2)2, 5-diphenyltetrazolium bromide) test (AU/100 million sptz), the centrifuged treatments resulted in values of 1.58 +/- 0.79 (T1) and 2.02 +/- 1.88 (T2), better than the treatment control (0.20 +/- 0.08). Likewise, the fertilization and hatching rates, respectly, were better for the T1 (76.68 +/- 15.22% and 54.62 +/- 17.39%) and T2 (69.85 +/- 17.85% and 50.87 +/- 20.59%) treatments, than the control treatment (40.03 +/- 11.67% and 24.14 +/- 13.97%). The comparison of differential centrifugation with density gradient centrifugation was carried out for the first time with cryopreserved milt of R. quelen. In summary, both centrifugation techniques proved to be efficient in obtaining a better portion of functional sperm from thawed milt. This is based on the high rates of embryonic survival and hatching obtained after both differential and density gradient separation.