A tibial tuberosity radiolucency is sometimes identified on lateral radiographs of canine stifle joints, however little is known about the cause or significance. The purpose of this study was to describe the prevalence, association with other stifle conditions, and histopathologic characteristics of tibial tuberosity radiolucencies in a group of dogs. Radiographs of all canine stifle joints over 5 years were evaluated. Presence or absence of a tibial tuberosity radiolucency was recorded by an observer who was unaware of clinical status. Patient signalment and presence of other stifle joint conditions were recorded from medical records. A tibial tuberosity radiolucency was found in 145/675 dogs (prevalence = 21.5%). Statistically significant associations were identified between tibial tuberosity radiolucency and stifle condition (P < 0.0001), breed size (P = 0.011), and younger age of presentation (P = 0.001), but not with gender (P = 0.513). Dogs with a tibial tuberosity radiolucency had higher odds of having a medial patellar luxation than dogs without (OR = 9.854, P < 0.0001, 95% CI 6.422–15.120). Dogs with a tibial tuberosity radiolucency had lower odds of having a cranial cruciate ligament rupture than dogs without (OR = 0.418, P < 0.0001, 95% CI 0.287–0.609). Four canine cadavers, two with normal stifles and two with tibial tuberosity radiolucencies, underwent radiographic, computed tomographic, and histologic examination of the stifles. Computed tomography revealed a hypoattenuating cortical defect in the lateral aspect of the proximal tibial tuberosity that corresponded histopathologically to a hyaline cartilage core. Findings indicated that the tibial tuberosity radiolucency may be due to a retained cartilage core and associated with medial patellar luxation in dogs.