Abstract Objective: To determine incidence and risk factors of post-sterilization hyphema in shelter cats. Animals studied: Retrospective medical record review of 1204 cats and prospective screening of 195 cats. Procedures: The study consisted of three parts: (a) Survey responses were collected from 20 veterinarians, who perform high-quality high-volume spay-neuter (HQHVSN) in both shelter and public clinic settings; (b) medical records of 1204 cats were analyzed retrospectively over a 14-month time period; and (c) ophthalmic examinations, including tonometry, were performed prospectively on 195 cats before and after sterilization surgery over 8 weeks. Results: Nine of 20 surveyed veterinarians reported having witnessed hyphema in cats following sterilization surgery. Retrospective review of 1204 medical record and prospective screening of 195 cats showed that three juvenile (<1 year of age) male cats (<2 kg) developed hyphema within 1 hour following surgery (0.2% incidence). In all three affected cats, anesthesia was induced with tiletamine/zolazepam (3 of 523 cats induced with this drug combination; 0.6% incidence), and hyphema resolved within 20 hours. Mean intraocular pressures as measured by Icare® TonoVet were (mean ± standard deviation) 11.5 ± 3.8 mm Hg and 21.7 ± 4.6 mm Hg for juvenile (<1 year of age) and adult (>1 year of age) cats, respectively. Conclusions: Survey responses and three observed cases confirm the existence of feline post-sterilization hyphema with an estimated incidence of 0.2%. The underlying mechanism for this occurrence remains unknown.