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Accurate measurement of testicular volume (TV) in boys is an important tool in clinical practice, e.g., in varicocele treatment. This study aims to assess the degree of intra- and interobserver variability of testicular volume measurements. In a prospective study, boys between 11 and 17 years of age without testicular pathology were enrolled. Testicular ultrasound was performed by three investigators (A: pediatric radiologist; B: pediatric surgery/urology resident; C: pediatric urologist). Intraobserver variability was calculated in investigators B and C and interobserver variability between all three investigators. A total of 30 boys were enrolled. Mean intraobserver variability in both observers was +0.3% with a range of −39.6 to 51.5%. The proportion of measurements with a difference >20% was 18.6%. The mean interobserver variability was −1.0% (range: −74.1% to 62.8%). The overall proportion of measurements with a difference >20% was 35%. A lower testicular size of < 4 mL showed a significantly higher rate of >20% difference in both the intraobserver group (31.1% vs. 14.4%; p = 0.035) and the interobserver group (63.2% vs. 26.2%; p = 0.000031). Furthermore, the rate of >20% difference was significantly lower in obese compared to non-obese patients in both the intraobserver (2.8% vs. 22.4%; p = 0.0084) and the interobserver group (24% vs. 40.8%, p = 0.0427). Both intraobserver and interobserver variability in ultrasound-based TV measurements in pubertal boys contain a relevant degree of uncertainty that renders them unsuitable for individualized follow-up care. At the cohort level, however, mean differences in ultrasound-based TV measurements are low enough to make ultrasound comparisons reasonable.