Evaluation of sex ratios is a critical component of chelonian captive breeding programs and may become increasingly useful to assess the demographics of free-living populations. In many reptile species, the sex of immature animals cannot be determined based on external features. Endoscopic sex identification is an accurate and safe method to identify the sex of immature individuals of some chelonian species. A number of studies describe this technique in controlled, hospital settings and report significant interspecies variations in gonad morphology; however, there are few reports describing this technique in field conditions. In the current study, the gonadal morphology of 40 immature Western Santa Cruz tortoises (Chelonoidis porteri) on Santa Cruz Island in Galapagos, Ecuador, was assessed. A previously described endoscopic protocol was used to perform sex identification under field conditions. Tortoises were anesthetized using an intramuscular injection of ketamine (10 mg/kg) and medetomidine (0.1 mg/kg), which provided an adequate plane of anesthesia. The medetomidine was reversed with atipamezole (0.5 mg/kg). Field conditions presented challenges such as limited control over lighting, suboptimal patient positioning, and restricted power supply for endoscopy equipment. The immature testicle in Western Santa Cruz tortoises was oval, reddish pink, and tightly adhered to the coelomic membrane ventral to the kidney. The surface of the gonads resembled other species with the notable exception that the ovaries lacked a significant number of primordial follicles. These gonadal characteristics were consistent, with only one individual identified as undetermined sex of the 40 samples. This field-based endoscopic gonadal evaluation was a safe and sensitive technique for determining the sex of free-living immature Western Santa Cruz Galapagos tortoises.