Aim: To investigate the validity of measurement of testicular volume acquired by a built-in software in different ultrasound systems with reference to the updated guidelines. Materials and methods: Archives of 1,976 patients who had undergone scrotal ultrasound evaluation were reviewed. A total of 973 patients with 1,909 testes, who had undergone ultrasound measurement of the testicular volume, were included in the study, and 1,003 patients were excluded. The age of enrolled patients ranged from 17 to 66 years (median age of 39 years). The ultrasound systems included Siemens Sonoline S2000, Philips EPIQ5, GE Logiq E9, Hitachi Aloka prosoundα7, Mindray DC-8 and Mindray Resona7. The transducers have imaging frequencies of 5–14 MHz. Validity of the measurement of testicular volume acquired by a built-in software in different ultrasound systems was assessed with reference to the formula that Volume (V) = Length (L) × Width (W) × Height (H) × 0.71, recommended by the updated guidelines, by recalculating the original numbers using a calculator. Results: The values obtained by the built-in software of Mindray DC-8 and Mindray Resona7 ultrasound systems and measurements recalculated on a computer were all in concordance; and the values obtained by the built-in software of Siemens Sonoline S2000, Philips EPIQ5, GE Logiq E9, and Hitachi Aloka prosoundα7 ultrasound systems and measurements recalculated on computer were all discordant. The same testicular measurements calculated with different formulas (V = L×W×H×0.71 vs. V = L×W×H×0.52) produced 26.76% difference. Conclusion: Values of testicular volume obtained by some ultrasound systems are not accurate with reference to the formula recommended by the updated guidelines.