In 2004, a subset of a translocated population of gopher tortoises (n = 14; Gopherus polyphemus) were captured to determine the overall health of individuals and to investigate the prevalence of selected pathogens. Additionally, ultrasonography and endoscopic visualization of the reproductive tract were compared as methodologies to accurately determine sex and reproductive status. Tortoises were premedicated with butorphanol and induced with propofol. The hematology and biochemical profile values obtained were within the reference ranges previously reported for gopher tortoises. Tortoises were parasitized with Alaeuris spp. of pinworms and trichostrongyles. Ten of the tortoises (71%) were also parasitized by hemogregarines. None of the animals had clinical signs indicative of acute upper respiratory disease (URTD); however, six animals had signs consistent with previous upper respiratory infections. One tortoise had an antibody titer against Mycoplasma agassizii, considered to be positive, while an additional five animals had antibody titers considered to be “suspect.” We failed to detect Mycoplasma sp., Salmonella sp., or Chlamydophila sp. DNA from any of the animals. In general, morphometric characteristics reliably predicted the correct sex in the majority of animals that were >15 yr of age. When compared to endoscopy, ultrasound examination of the gonads was accurate for sex determination in 57% of the tortoises examined and was 86% accurate in animals >15 yr of age. Ultimately, endoscopy provided the most accurate method for determining sex in chelonians, particularly in juveniles or during nonreproductive seasons.