The objective of this study was to compare surface, esophageal, and cloacal temperatures in awake iguanas, chameleons, and snakes at two different ambient temperatures and in alligators at one ambient temperature. Surface, esophageal, and cloacal temperatures were measured in all animals twice, with exception of the alligators, where temperatures were taken once. The first set of temperature readings was done at lower environmental temperature. An analysis of variance (ANOVA) with a mixed-effect model was used to analyze the temperature difference between esophageal and cloacal temperature against each other, and against surface temperature. Significance was set at P < 0.05. In all animals, surface, esophageal, and cloacal temperatures increased significantly (P < 0.0001) with increased ambient temperature. Esophageal and cloacal temperature were not significantly different from each other in iguanas and chameleons at lower environmental temperature or in snakes and chameleons at high ambient temperature. In snakes, esophageal temperature 26.1 ± 1.6°C was significantly higher than cloacal temperature 25.2 ± 0.9°C (P = 0.0016) at lower ambient temperature. In alligators no difference between esophageal and cloacal temperature was observed at the given ambient temperature. Surface temperature was significantly lower than esophageal and cloacal temperature in all species investigated, except iguanas at lower ambient temperature. The results of this study suggest that in healthy awake iguanas and chameleons at any ambient temperature and in snakes at high ambient temperature, cloacal temperatures are reflective of esophageal temperature in the species evaluated. In alligators, rectal temperature reflected esophageal temperature at the given ambient temperature. Surface temperature in contrast was underestimating esophageal temperature in all species investigated.