Cryopreservation of spermatozoa from free-living ocelots (Leopardus pardalis) could benefit their conservation by facilitating gene flow between in situ and ex situ populations without requiring removal of additional cats from the wild. The objective of this study was to investigate three different methods of ocelot sperm cryopreservation to identify the most appropriate technique for use in a field environment. Male ocelots (n = 10), housed in North American zoos, were anaesthetised with tiletamine–zolazepam (7mg kg^–1 bodyweight; i.m.) and subjected to a regimented electroejaculation procedure. Recovered semen was evaluated for sperm concentration, motility and morphology and processed for cryopreservation by three methods: (1) pelleting on dry ice, (2) freezing in straws over liquid nitrogen vapour; and (3) freezing in straws in a dry shipper. Frozen samples were thawed and assessed for post-thaw acrosome status, viability, motility over time and ability to fertilise viable domestic cat oocytes. Although several post-thaw sperm parameters varied (P < 0.05) among freezing methods, frozen–thawed ocelot spermatozoa from all treatments showed a similar (P > 0.05) capacity to bind, penetrate and fertilise viable domestic cat oocytes. These findings suggest that spermatozoa collected from male ocelots under field conditions may be frozen in straws either using liquid nitrogen alone or in a charged dry shipper to retain adequate functional competence after thawing for use with assisted reproductive procedures.