This study compared three different techniques for sperm cryopreservation of Tambaqui (Colossoma macropomum). Semen was diluted in Beltsville Thawing Solution with the addition of dimethyl sulfoxide (DMSO) at various concentrations (5%, 10%, 15% and 20%). Cryopreservation was performed using three methods: Box Conditioner Method with straws at a 5 cm distance from liquid nitrogen vapor (N2L); Dry Shipper Method placing the straws inside the machine; Vitrification Method placing the straws directly into N2L, amounting to 12 treatments (four DMSO concentrations×three freezing methods). The samples were evaluated for analysis of sperm quality in vivo and in vitro. Use of the Vitrification Method at different concentrations of DMSO provided the least values in the different evaluations. Fertilization, hatching rates and plasma membrane integrity using the Box Conditioner Method with 5% and 10% DMSO did not differ (P>0.05) but use of the concentration of 5% DMSO resulted in greater values than the other treatments (P<0.05) as well as for sperm motility and latency time (P<0.05), although sperm viability was superior using the Dry Shipper Method with 20% of the cryoprotectant. Mitochondrial functionality was impaired by use of the Vitrification Method with all DMSO concentration tested showing the most desirable values when the Box Conditioner Method was used with 5%, 10%, 15% DMSO and the Dry Shipper Method was used with 10% and 15% DMSO. Considering the variables evaluated, the use of the Box Conditioner Method is associated with enhanced Tambaqui semen quality with freeze concentrations of 5% and 10% DMSO.