The aim of the present study is to determine the basal parameters in opossums (Didelphis virginiana) during physical examination in Yucatan, Mexico. Sixty-six opossums were captured and manually handled for physical examinations. Sex, age, cardiac and respiratory rate, body temperature, pulse, mucous membranes, capillary refill time, hydration, size of superficial lymph nodes, mental status, and body condition were evaluated. Also, comparisons between rectal and auricular temperatures were performed. The means of physiological parameters obtained were: cardiac frequency 146 beats per minute (95% confidence interval [CI]: 138.91–155.24), respiratory frequency 27.33 breaths per minute (95% CI: 25.15–29.64), and body temperature 34.01°C (95% CI: 33.71–34.31). In 54% of animals, a sinus arrhythmia was present. A significant difference was found between rectal and auricular temperature measurements. A variation of −1.64 to 1.14°C was obtained via the auricular thermometer compared with the rectal device.