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Gnotobiotic piglets (n= 10) were hand-reared in conventional germ-free facilities. Piglet body temperatures were measured with rectal and non-contact infrared thermometry (NIFT) on the lower eyelid, auricular center and margin, parietal regions, axilla, central abdomen and dorsum, and the perianal region. Body temperature measurements at central abdomen, cranial dorsum, and perianal regions had NIFT values which had a significant linear relationship (p< 0.0001) with rectal thermometry. The predicted equations of between-subject formulas were calculated as follows: rectal temperature, 28.07489+ 0.30372 central abdominal surface temperature; rectal temperature, 34.02799+ 0.15197 central dorsum surface temperature; and rectal temperature, 33.87937+ 0.15676 perianal temperature. These results suggested that NIFT could serve as a valid alternative to rectal thermometry in a portable germ-free facility without disturbing experimental animals. The development of a NIFT body temperature evaluation that does not require animal restraint is clinically advantageous, particularly in gnotobiotic piglets, and would be significantly less stressful for experimental procedures in germ-free facilities.