Assisted reproductive technologies provide important tools for wildlife conservation but have rarely been developed for reptiles. A critical step in developing cryopreservation protocols is establishing optimal cooling rates for cell survival. The two-factor hypothesis explaining cryoinjury to cells originates from an inverted 'U' shape of recovery curves generated in many cell types thawed after cryopreservation, due to cell recovery declining at cooling rates either side of a single optimum. We generated such a curve for the yellow-spotted monitor lizard Varanus panoptes, the first for any reptile. We cryopreserved sperm using two cooling devices (LN2 dry shipper; LN2 bath vapour) and two sperm-holding vessels (Cassou sperm straws; Nunc CryoTubes) to generate four different cooling-rate curves during freezing. Sperm motility and viability (47.3 % and 76.5 % respectively) was highest when frozen in straws suspended in a LN2 bath at an intermediate cooling rate of 73oC/min between 0 and -50oC, whereas sperm frozen in straws suspended in a dry shipper at the fastest cooling rate (231oC/min between 0 and -50oC) produced the lowest recovery (10.4 % and 36.4 % motility and viability, respectively). Sperm frozen in cryotubes at the lowest cooling rates in either LN2 bath vapour or dry shipper produced intermediate recovery. The shape of the optimal cooling curve conformed to the two-factor hypothesis of cryoinjury, the first such evidence in reptile sperm. This in turn led to the identification of simple cryopreservation setups (LN2 vapour with straws and cryotubes; dry shipper with cryotubes but not straws) suitable for cryopreserving lizard sperm in the field.