The desert tortoise (Gopherus agassizzii) is listed as threatened by the USA Fish and Wildlife Service and population declines continue to occur throughout most of their range. This species’ low reproductive rate, combined with the advanced age at which they reach sexual maturity, makes them vulnerable to multiple threats. Although assisted reproductive technologies can enhance breeding of many species, they are not widely used in tortoises. The objective of this study was to identify effective sperm cryopreservation protocols for the desert tortoise and possibly to other members of Testudinidae. We compared the effects of various concentrations of the cryoprotectants dimethyl sulfoxide (DMSO) and glycerol (6–20%) using 3 freezing devices at 4 freeze rates (CryoCooler, Ops Diagnostics, 2.3°C/m, 6.4°C/m; CryoMed, Thermo Scientific, 0.3°C/m or 1.0°C/m; and CoolCell, Biocision, 1.0°C/m) on several sperm parameters. Sperm was collected postmortem from the vas deferens of 9 individuals and tested either individually (n = 2), combined into 1 pool of 3 individuals, or 2 pools of 2 individuals. Sperm was extended in TEST-yolk buffer. Initial motility score (IMS; % motile × speed of progression²), plasma membrane integrity (IPL), and acrosome integrity (IAC) were recorded before cryoprotectant addition and freezing. For each treatment group, triplicate vials were thawed at 37°C for 60 s. Cryoprotectant was removed by centrifugation and the sperm pellet was resuspended in M199 + HEPES. Sperm were evaluated immediately following resuspension (T0), as well as 30 (T30) and 60 (T60) minutes postincubation at 22°C. All data were expressed as a percentage of initial (%IMS, %IPL, and %IAC). A sperm quality index (SQI) was calculated as (%IMS × %IPL × %IAC)/1,000, giving equal weight to each indicator of cryosurvival. The effects of freeze method on %IMS, %IPL, %IAC, and SQI were analysed by ANOVA and Tukey’s test. The effect of freeze method was significant at T0 and T60, with the 16% DMSO, 6.4°C/m method resulting in the highest %IMS at T0 (46.1%) and T30 (33.8%) and the 12% glyercol at 0.3°C/m highest at T60 (48.7%). Sperm frozen in 16% glycerol at 0.3°C/m had the highest %IPL at T0, T30, and T60 (91.9, 90.4, and 85.4%, respectively). Acrosome integrity was best maintained when sperm were frozen in 16% DMSO at 6.4°C/min (91.9%). The SQI was highest at T0 when sperm was frozen at 1.0°C/min in the CryoMed with highest post-thaw sperm parameters in 16% (T0) or 12% glycerol (T30 and T60). Interestingly, there were significant differences in SQI between the two 1.0°C/min freeze methods at each period, indicating that freezing device affected sperm cryosurvival, perhaps due to different freezing curves. This study indicates that mid-range (12 and 16%) cryoprotectant concentrations and slow freeze rates (0.3°C/m and 1.0°C/m) are optimal for desert tortoise sperm frozen in TEST-yolk buffer. Future studies will determine fertilizing capability of these sperm. These results may serve as a starting point for the study of sperm cryopreservation in other Testudinidae species.