Coccidioidomycosis, or Valley Fever, is a fungal infection caused by Coccidioides spp., soil-living fungi endemic to the southwest region of the United States. The infection can cause various diseases including respiratory, neurologic, cardiac, dermatologic, and ocular disease as well as osteomyelitis in dogs and many other mammals. Obtaining a definitive diagnosis can be challenging. Serology is commonly used as a screening diagnostic test for disease, but both false-negative and false-positive results have been reported. Fifty-two cases of coccidioidomycosis diagnosed via histopathology were retrospectively evaluated. The sensitivity of serology in the study population was determined to be 87% for immunoglobulin G and 46% for immunoglobulin M. The cases were evaluated for an association between negative serology results and anatomic location of disease, but these variables were found to be independent. This study reports the sensitivity of serology for canine coccidioidomycosis and highlights the importance of using multiple diagnostic tests for definitive diagnosis of infection.