A reliable method for obtaining renal ultrasonographic measurements in the horse is important for diagnosis and monitoring of clinical renal disease. The aims of this prospective study were to develop and validate a novel translumbar ultrasound technique for measuring renal dimensions in horses. Six Thoroughbred or Thoroughbred part bred horses were recruited. All horses were scheduled for euthanasia due to reasons unrelated to the kidneys. Two observers recorded renal length, width, and depth; and dimensions of the cortex, medulla, pyramids, and pelvis for both kidneys in each horse using novel translumbar and conventional transabdominal ultrasound methods. The same measurements were recorded from post-mortem renal specimens. Both kidneys were consistently identified by both methods in the 15–17th intercostal spaces and paralumbar fossa. Using the translumbar technique, maximal dimensions were obtained for the left kidney in the 16th intercostal space (length 16.2 ± 2.0 cm, width 11.8 ± 0.5 cm, depth 6.4 ± 0.9 cm) and for the right kidney in the 15th intercostal space (length 16.1 ± 1.2 cm, width 13.4 ± 1.2 cm, depth 6.7 ± 0.7 cm). Renal dimensions obtained by transabdominal and translumbar projections did not differ (P > 0.05). Good correlations were found between overall renal dimensions and post-mortem measurements for both ultrasound techniques (r2 > 0.8), but were better for the translumbar method (mean r2 = 0.92 cf. 0.88). Good-to-excellent reliability was found for all translumbar ultrasound measurements except for the renal cortex. Reproducibility was better for the larger (overall length, width, and depth) than the smaller (cortex, medulla, and pyramids) structures. Findings indicated that translumbar ultrasonography is a valid method for measuring renal dimensions in horses.