Evidence-informed practice is currently lacking in canine hydrotherapy. This study aimed to investigate if the estimated workload of the gluteus medius (GM) and longissimus dorsi (LD) increased in dogs at different water depths when walking on a water treadmill. Seven dogs were walked for 2 min continuously on a water treadmill at depths of no submersion (depth 1), mid-tarsal (depth 2), between lateral malleolus and lateral epicondyle (depth 3) and between the lateral epicondyle and greater trochanter (depth 4). Continuous electromyographic data from the right and left sides of GM and LD were collected simultaneously during exercise. Friedman’s analyses with post-hoc Wilcoxon tests established if significant differences in GM and LD muscle activity occurred between the water depths for mean estimated-workload. Significant differences occurred in estimated-workload in GM and LD between water depths (P<0.05). Mean estimated-workload decreased in the right and left GM between depths 2 (mid-tarsal) and 3 (between lateral malleolus and epicondyle) (P<0.007) and depths 2 and 4 (between lateral epicondyle and greater trochanter) (P<0.001), a pattern which was repeated for left and right LD (P<0.007). Right GM mean estimated-workload increased between depth 1 (no submersion) and depth 2 only (P<0.013). Water depth influences GM and LD activity in dogs walking on a water treadmill. Increasing knowledge of canine locomotion in water treadmills could be used to inform individualised rehabilitation regimes for dogs undertaking hydrotherapy.