An increasing proportion of canine patients are presented with chronic thoracolumbar back pain and without compressive spinal lesions. In humans, spinal perineural infiltrations have been reported to have a favorable effect on pain control. The purpose of this prospective cadaver study was to describe the dispersal pattern of injectate following CT-guided spinal perineural infiltration in the canine thoracolumbar region. Seven fresh canine cadavers were first scanned using multislice CT and then CT-guided spinal perineural infiltration was performed at 42 sites from T13/L1 to L6/L7. The injectate for each site was a mixture of new methylene blue and iodinated contrast medium. Immediately following CT-guided injection, cadavers were frozen, cut, and dissected macro- and mesoscopically (using a magnifying glass) to identify anatomic structures that were infiltrated. In the majority of sites (64.3%), complete epidural and hypaxial staining of spinal nerve components (including the spinal ganglion, trunk, and ventral branch) was successfully achieved. However, no (11.9%) or unpredictable staining (9.5%) of nervous tissue occurred in some sites despite careful CT guidance and the application of relatively large volumes of injectate. Optimal results were achieved when the needle tip was positioned periforaminally ventral to the cranial contour of the cranial articular process. Findings from this ex vivo study indicated that CT-guided spinal perineural infiltration is feasible for testing in the canine thoracolumbar region and that successful nerve tissue infiltration would likely occur in the majority of sites. Future in vivo studies are needed to determine the safety and efficacy of this technique.