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To determine whether canine ovariohysterectomy or orchiectomy affects the prevalence of anterior cruciate ligament injury, we compared injury rates of anterior cruciate ligaments of animals that had gonadectomy and animals that were sexually intact as a function of gender, breed, or size. Records of 3218 dogs treated in one orthopaedic veterinary practice during a 2-year period were retrospectively reviewed. Anterior cruciate ligament injury, diagnosed by a history of acute hind limb lameness and by positive anterior drawer test, was confirmed at the time of surgery. The prevalence of anterior cruciate ligament rupture in all dogs was 3.48%. Females that had ovariohysterectomy and males that had orchiectomy had a significantly higher prevalence of anterior cruciate ligament rupture than the sexually intact dogs. Larger dogs had an increased prevalence of anterior cruciate ligament injury compared with smaller or medium-sized dogs, with the increased rupture rates for sterilized animals holding across breeds and sizes. Sterilization of either gender increased the prevalence of anterior cruciate ligament injury, suggesting a potential effect of gonadal gender on prevalence of injury of this ligament.