Cold-stress syndrome (CSS) is one of the leading natural threats to free-ranging Florida manatees (Trichechus manatus latirostris). Cold water exposure below the species' acceptable physiologic range is a frequent occurrence for manatees during cold weather months causing CSS-induced systemic illness and significant annual mortality. Although CSS is a commonly presented condition at manatee rehabilitation facilities, the core body temperatures in CSS manatees are currently unknown due to the lack of clinically applicable and accurate temperature measurement methodologies. Our objective was to establish a clinically applicable measurement methodology of core body temperature in manatees. A novel, minimally invasive temperature technique to obtain esophageal temperature by placing a temperature sensor through an oro-gastric tube was compared to current oral and nasal methods in 20 clinically healthy manatees. Results identified the esophageal measurement as the best performing and most precise temperature methodology. The superior performance of esophageal temperature measurements differed significantly from both nasal and oral measurements, while nasal and oral measurements did not differ when compared with each other. The esophageal measurements were consistent with manatee core body temperature, facilitating generation of a reference interval for core body temperature in healthy manatees (35.0–35.8 C). Four CSS medical cases were evaluated with the newly validated esophageal temperature method, facilitating diagnosis of hypothermia. The application of this temperature measurement technique to CSS manatees in field or rehabilitation settings will help in understanding CSS pathophysiology, improve medical assessments during rehabilitation, and contribute to conservation efforts.