The purpose of this study was to determine if variation in the ultrasound beam angle would affect cartilage thickness measurement performed with B-mode ultrasonography. Transverse sections of six fresh equine middle phalanges were obtained from necropsy. Ultrasonographic images of the proximal articular cartilage were obtained in a water bath, in a plane parallel and adjacent to the section plane using a 5–10 MHz linear transducer. Static images were acquired for all six bone specimens with an ultrasound beam angle of 0°, 30°, 45°, and 60°. Proximal articular cartilage thickness was measured on ultrasonographic images and on the bone specimen at the same level. A linear mixed-effects model was used to compare articular cartilage thickness measured on specimen and on ultrasonographic images using different ultrasound beam angle. Mean±SD cartilage thickness was 1.82±0.35 mm on bone specimens, 1.72±0.29 with a 0° angle, 1.99±0.34 with 30°, 2.06±0.34 with 45°, and 2.3±0.38 with 60°. There was a significant difference between macroscopic measurements and ultrasonographic measurements performed with ultrasound angles at 30°, 45°, and 60°. There was a significant increase in cartilage thickness when the ultrasound beam angle decreased (P=0.0157; R2=0.969). Cartilage thickeness measured on ultrasonographic images varies with the ultrasound beam angle and may not be accurate because ultrasound speed in cartilage may be different than the speed used by the ultrasonographic unit for distance calculation.