Objective There is a risk of biological contamination from egg yolk and unidentified components in traditional sperm cryopreservation solution. Therefore, this study aimed to use two commercial yolk-free cryopreservation solutions, Origio and Quinn’s, to preserve the semen of cynomolgus monkey and to determine the optimal cryopreservation conditions for this species. Methods Semen from male monkeys was collected by penile electrical stimulation. The two kinds of cryopreservation solution were used to freeze sperm under conditions of different freezing suspension heights above the surface of liquid nitrogen and different cryopreservation liquid volume ratios. The optimal conditions of cryopreservation solution were determined from sperm viability, recovery and acrosomal integrity rates after cryopreservation, and the effect of seminal plasma on sperm cryopreservation was confirmed using optimal conditions with Quinn’s cryopreservation solution. Results The optimal conditions for cryopreservation of cynomolgus monkey sperm with Origio and Quinn’ s solution were suspended at 5 cm above liquid nitrogen, and a volume ratio of semen to cryopreservation solution of 1 ∶0. 5. Under these optimal conditions, the recovery rate using Quinn’ s solution 38. 02% ± 14. 98% was significantly higher than that using Origio solution 15. 11% ± 14. 49%. There was no significant difference in acrosomal integrity between the two solutions. Preservation using seminal plasma can improve the freezing effect of yolk-free cryopreservation solution. The recovery and acrosomal integrity rates with seminal plasma were 37. 57% ± 13. 22% and 84. 64% ± 8. 82%, respectively, compared with recovery and acrosomal integrity rates without seminal plasma of 21. 46% ± 7. 25% and 75. 50% ± 9. 62%, respectively. Conclusions Yolk-free cryopreservation solution successfully preserved cynomolgus monkey sperm, and provides a safe and reliable way for conserving the fertility of male cynomolgus monkeys.