The aim of this study was to determine if infrared thermography can be used as a tool to detect jugular venipuncture in the horse to support United States Equestrian Federation drug administration rule enforcement. This is a blinded, randomized clinical trial, and eight adult Thoroughbreds were used. For each horse, thermographic images of both jugular grooves were obtained by a blinded examiner, prior and subsequent to jugular venipuncture (10-mL sterile saline). Injected sides were randomized, and images were acquired before injection and then 0.5 hours, 1 hour, 2 hours, 3 hours, 6 hours, 12 hours, 24 hours, and 36 hours after injection. Horses were maintained in a temperature-regulated stall. Images were reviewed and analyzed with standard thermography software to determine if venipuncture could be detected. Images of treated sites were minimally visibly different from those obtained at baseline or for the control sites. Following image analysis, a significant reduction (P < .05) was identified when mean jugular groove injection site temperature measurements were compared to baseline mean temperature measurements. The greatest observed difference (1.4 ± 0.8°C) was identified at 2 hours after injection. At time points 0.5 hours, 6 hours, and 12 hours, delta T (difference between the mean temperature at each time point and the baseline temperature) was significantly different between the jugular injection site and the control site. Infrared thermography detects a significant change in surface skin temperature from baseline following jugular venipuncture for sterile saline injection. No significant comparable observed changes were identified at the control sites.