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This study aimed to evaluate various concentrations of egg yolk (5, 10, or 20%) in combination with different concentrations of glycerol (3% or 6%) added to a Tris-based extender on the post-thaw characteristics of sperm obtained from Tayassu tajacu. For this purpose, semen from 10 sexually male mature collared peccaries was collected by electroejaculation and evaluated for sperm motility, vigour, viability, morphology and functional membrane integrity. The ejaculates were initially extended in Tris-fructose plus egg yolk (5%, 10% or 20%). After cooling, the semen was added to Tris-egg yolk plus glycerol (6% or 12%), resulting in a final concentration of 3% or 6% glycerol of the extender. Straws were frozen using liquid nitrogen and thawed in a water bath at 37°C for 30 s. The frozen–thawed semen was evaluated as reported for fresh semen. After thawing, a significant decrease was verified for sperm motility and vigour, for all the samples in comparison with fresh semen. However, no differences were evidenced among treatments for any sperm characteristics evaluated (p > 0.05), except for the combination between 10% egg yolk and 6% glycerol, which provided the worst preservation of functional membrane integrity (p < 0.05). The interactions between higher concentrations of egg yolk (20%) and glycerol (6%) and also between lower concentrations of the same substances (5% egg yolk and 3% glycerol) added to the Tris-based extender negatively affected the preservation of the normal sperm morphology after thawing (p < 0.05). In conclusion, the use of Tris-based extender added to 10% or 20% egg yolk plus 3% glycerol is recommended for effective sperm cryopreservation in collared peccaries.