Alpha₂-adrenergic agonists are used to immobilize many veterinary species, but use has been infrequently linked to urine contamination of semen collected via electroejaculation. The objective of the study was to compare the α₂-agonists medetomidine and dexmedetomidine on urine contamination of semen in anesthetized cheetahs (Acinonyx jubatus) during electroejaculation procedures. From 2009–2012, a retrospective medical record review revealed 21 anesthesia events in 12 adult male cheetahs. Animals were immobilized with combinations of Telazol® (2.33 ± 0.43 mg/kg) and ketamine (2.38 ± 1 mg/kg); Telazol (1.17 ± 0.14 mg/kg), ketamine (1.17 ± 0.14 mg/kg), and medetomidine (0.012 ± 0.0017 mg/kg); or Telazol (1.59 ± 0.1 mg/kg), ketamine (1.59 ± 0.1 mg/kg) and dexmedetomidine (0.01 ± 0.001 mg/kg). Semen was successfully collected in all animals; four animals anesthetized with medetomidine had urine contamination (P = 0.037). Medetomidine may contribute to urine contamination; however, further investigation is needed to determine significance in cheetahs.