This study was undertaken to evaluate the reliability of two-dimensional (2D) ultrasound in measuring testis size in dolphins, in vivo, with the subject presenting for examination under voluntary or trained behaviour. The testes of five bottlenose dolphins (Tursiops aduncus) were measured once by two operators to test inter-operator variability (reproducibility) and repeatedly measured by the same operator to test intra-operator variability (repeatability). Ultrasound examinations for each test were conducted on the same day to avoid measurement variability due to time difference. The evaluation of reproducibility and repeatability were conducted on separate days. In the ultrasound examination, the length, circumference, depth and width of both testes of the animal were measured. To prevent bias, measurements were not communicated between the operators on-site and repeated measurements were masked. Results showed that both reproducibility and repeatability of all the testis measurements were high (>90%). Overall, measurement variability of the technique was found to be of a satisfactory level. Ultrasound is a useful imaging tool for routine long-term monitoring of the testes in this species of animals. Sources of error due to movements as a result of the subject being in the water during examinations were inevitable and must be taken into account. (E-mail: htqueeny@polyu.edu.hk). ; Department of Health Technology and Informatics