Tracheal collapse in horses is reportedly uncommon; however, American Miniature Horses are more commonly affected. There is no description of the tracheal luminal diameter of American Miniature Horses, making early detection of tracheal luminal narrowing difficult. To 1) describe radiographic tracheal luminal diameter in clinically normal American Miniature Horses, 2) report the prevalence of subclinical tracheal collapse in a population of American Miniature Horses, and 3) use tracheal videofluoroscopy to quantify variation in tracheal luminal diameter throughout the respiratory cycle in horses with no clinical respiratory disease. Descriptive observational reference interval study. Thirty-four American Miniature Horses with no reported history of respiratory illness were recruited. Lateral cervical and thoracic radiographs were obtained in unsedated standing horses. Dynamic fluoroscopic images were obtained of the cervical and thoracic trachea throughout the respiratory cycle. Horses were then sedated as needed and tracheoscopy was performed. Twenty-nine horses were categorised as normal, and five horses were categorised as subclinically affected based on 25% or greater tracheal narrowing using tracheoscopy for visual assessment. Radiographic tracheal lumen to vertebral body measurements were obtained throughout the cervical and thoracic trachea. Maximum and minimum fluoroscopic tracheal diameter at each site throughout the respiratory cycle was recorded. A mean, median, 95% confidence interval and bootstrapped 95% reference interval of radiographic tracheal diameter to vertebral body ratios were generated in normal horses. The prevalence of subclinical tracheal collapse in this population of American Miniature Horses is 14.7%. Bootstrapped reference range was generated from 29 horses. Radiographic tracheal measurements and ratios of the tracheal diameter to vertebral body in the normal American Miniature Horse are described herein and can be used as a guideline when screening for tracheal disease in American Miniature Horses. The prevalence of subclinical tracheal collapse in American Miniature Horses may be higher than previously reported. © 2019 EVJ Ltd.