Subarachnoid lumbar puncture is used commonly in the dog for cerebrospinal fluid collection and/or myelography. Here in we describe the percutaneous ultrasound anatomy of the lumbar region in the dog and a technique for ultrasound-guided lumbar puncture. Ultrasound images obtained ex vivo and in vivo were compared with anatomic sections and used to identify the landmarks for ultrasound-guided lumbar puncture. The ultrasound-guided procedure was established in cadavers and then applied in vivo in eight dogs. The anatomic landmarks for the ultrasound-guided puncture, which should be identified on the parasagittal oblique ultrasound image are the articular processes of the fifth and sixth lumbar vertebrae and the interarcuate space. The spinal needle is directed under ultrasound-guidance toward the triangular space located between the contiguous articular processes of the fifth and sixth lumbar vertebrae and then advanced to enter the vertebral canal. Using these precise ultrasound anatomic landmarks, an ultrasound-guided technique for lumbar puncture is applicable in the dog.