Epilepsy, or recurrent seizures, is reported to be the most common neurologic condition in dogs; 20–30% of epileptic dogs are considered to be pharmacoresistent to one medication. The hormone melatonin has been shown to have significant anticonvulsant effects; epileptic humans have lower serum melatonin levels than unaffected individuals. We hypothesize that serum levels of melatonin will be lower in dogs with seizures as compared with normal dogs. Sixty-two dogs were enrolled in the study: 29 normal dogs (Group 1) and 33 dogs with seizures (Group 2). Blood sampling was done at three separate time points (8:00 a.m., 12:00, and 4:00 p.m.). The majority of dogs in Groups 1 (69%) and 2 (76%) had serum melatonin levels <0.5 pg/mL as measured by radioimmunoassay. There were no significant differences in serum melatonin values between the groups or within groups when time of blood draw, length of sample freezing, time of day/year of sampling, or presence of anticonvulsant therapy were compared. There were no notable differences in daytime serum melatonin values in normal dogs versus dogs with seizures. The majority of daytime serum melatonin levels were <0.5 pg/mL in dogs with and without seizures.