During the last decade, a number of pain assessment tools based on facial expressions have been developed for horses. While all tools focus on moveable facial muscles related to the ears, eyes, nostrils, lips, and chin, results are difficult to compare due to differences in the research conditions, descriptions and methodologies. We used a Facial Action Coding System (FACS) modified for horses (EquiFACS) to code and analyse video recordings of acute short-term experimental pain (n = 6) and clinical cases expected to be in pain or without pain (n = 21). Statistical methods for analyses were a frequency based method adapted from human FACS approaches, and a novel method based on co-occurrence of facial actions in time slots of varying lengths. We describe for the first time changes in facial expressions using EquiFACS in video of horses with pain. The ear rotator (EAD104), nostril dilation (AD38) and lower face behaviours, particularly chin raiser (AU17), were found to be important pain indicators. The inner brow raiser (AU101) and eye white increase (AD1) had less consistent results across experimental and clinical data. Frequency statistics identified AUs, EADs and ADs that corresponded well to anatomical regions and facial expressions identified by previous horse pain research. The co-occurrence based method additionally identified lower face behaviors that were pain specific, but not frequent, and showed better generalization between experimental and clinical data. In particular, chewing (AD81) was found to be indicative of pain. Lastly, we identified increased frequency of half blink (AU47) as a new indicator of pain in the horses of this study.