In recent years, genome banks have grown as a way of maintaining the genetic variability of populations. However, the quality of gamete cryopreservation will determine their efficiency. This study aimed to evaluate prefreeze and postthaw sperm motility, vigor, membrane integrity, and morphology of semen of red brocket deer (Mazama americana) using three extenders: E1, Tris-Yolk; E2, Tes-Tris-Yolk; and E3, Tes-Tris-Yolk-Equex®. Six bucks were used, and three collections per buck were performed at 90-day intervals. Before freezing, semen volume, ejaculate concentration, motility, vigor, membrane integrity, and sperm morphology were evaluated. To compare the effect exerted by the extenders after sample thawing, further analyses of sperm motility, vigor, membrane integrity, and morphology were performed. Mean ejaculate volume and sperm concentration were 365.33 ± 120.5 μl and 2,675.73 ± 810.4 sperm/ml, respectively. Prefreeze motility for the extenders showed no significant differences (∼60%). Postthaw motility (E1 = 16.33 ± 5.5, E2 = 5.44 ± 5.2, E3 = 24.66 ± 10.0) was significantly different between E2 and E3, whereas postthaw vigor (E1 = 2.66 ± 0.8, E2 = 1.89 ± 1.2, E3 = 3.83 ± 0.4) was greater for E3 (P ≤ 0.05). Analysis of postthaw membrane integrity revealed no significant differences between the extenders regarding counts of cells presenting intact membranes; however, E3 promoted the lowest number of cells with damaged membranes and higher cell counts for partially damaged membranes (P ≤ 0.05). Analysis of sperm morphology revealed an increase in severe abnormalities when using E2 and E3 (P < 0.05). However, observation verified that counts of altered cells were lower using E3 than E2, suggesting a protective effect of Equex. These findings indicate that E3 promoted better semen quality postthaw. However, the performance of this extender in protecting sperm cells of M. americana during freezing was lower than that verified for other species. Thus, further studies are needed for characterization of red brocket deer semen and the optimization of extender for sperm cryopreservation.