Monitoring growth of neonatal dairy calves is a useful management tool to assist producers in achieving goals for reproduction and performance. The goal of this study was to examine ultrasound as an in vivo tool to quantify longissimus dorsi muscle (ribeye) linear depth and extensor carpi radialis (ECR) and semitendinosus (ST) muscle cross-sectional areas in postmortem preweaned Holstein calves. Postmortem preweaned calves (n = 137, age 13.1 d ± 15.5 SD, body weight 36.5 kg ± 7.2 SD) were obtained from two California calf ranches between April and July 2013. Two operators collected ultrasound images of the ribeye, ECR, and ST muscles using an Aloka 500V equipped with a 5-cm 7.5-MHz linear transducer. Ultrasound ribeye linear depth and ECR and ST cross-sectional areas were calculated using the Ultrasound Image Capture System. Ultrasound measurements were compared to dissected (carcass) measures. Carcass ribeye linear depth was estimated using a ruler. Dissected ECR and ST muscle cross-sectional areas were estimated by tracing muscle cross sections onto transparency paper and then photocopying, cutting out, and weighing individual paper muscle tracings. Weights of the tracings were then converted to areas using the known area of a standard 8.5 × 11 inch paper. Data were analyzed by regressing carcass estimates on observed ultrasound measurements. The coefficient of determination (R 2) indicated that ultrasound measurements were most closely associated with carcass measurements for the ST muscle (R 2 = 0.60, 0.62 for operator 1 and 2, respectively) when compared to the ribeye and ECR muscles (R 2 = 0.27, 0.41 for ribeye and 0.43, 0.32 for ECR for operator 1 and 2, respectively). The mean bias showed consistent underestimation by the ultrasound measurements when predicting carcass measurements for all three muscles and for both operators (ribeye bias = 0.15, 0.40; ECR bias = 0.95, 1.15; and ST bias = 0.73, 0.27 for operator 1 and 2, respectively). Operator contributed significantly in explaining a proportion of the variation in the regression equation for the ST muscle only, whereas calf body weight contributed significantly in explaining a proportion of the variation in the regression equation for all three muscles. The results of this study demonstrated that ultrasound measurements of the ST were the most accurate for quantifying the cross-sectional area when compared to both the ECR and ribeye in postmortem Holstein calves.