Successful sex identification in reptiles can often be challenging. The objective of this masterclass is to review techniques and clinical tools available to help clinicians successfully differentiate between male and female reptiles. Basic anatomy and physiology will be discussed including underlying reproductive tract embryogenesis, gonadal development, gonadal differentiation, and the known mechanisms behind sex differentiation in reptiles. Diagnostic methods for sex identification for immature and mature reptiles will be reviewed including sexual dimorphism, radiography, ultrasonography, computed tomography, plasma or fecal hormone analysis, and novel molecular methods.