OBJECTIVE To assess the relationship between plasma and RBC fatty acid composition and incidence and severity of squamous gastric ulcers when altered by short-chain (SC) or long-chain (LC) polyunsaturated fatty acid (PUFA) supplementation. ANIMALS 13 fit Thoroughbred horses in training. PROCEDURES Horses were evaluated by gastroscopy for squamous ulcer score, gastric pH, and blood fatty acid composition prior to supplementation (UNSUPP) and after 3 months of supplementation with a corn-flax oil blend of alpha-linolenic acid and linoleic acid (SC-PUFA) or a gamma-linolenic acid (GLA)-fish oil blend of GLA, eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA; LC-PUFA) in a crossover design. Prior to gastroscopy and blood collection, horses performed a 4,600-m standardized exercise test on the racetrack as a stressor. RESULTS Three months of supplementation with LC-PUFAs increased RBC levels of GLA, dihomo-gamma-linolenic acid (DGLA), arachidonic acid (AA), EPA, and DHA, and reduced severe ulcer prevalence (38% UNSUPP vs 8% LC-PUFA with a severe ulcer score of grade 3 to 4). Short-chain PUFA supplementation did not effectively elevate RBC GLA, DGLA, AA, EPA, or DHA and severe ulcer incidence was not different (38% UNSUPP vs 23% SC-PUFA with a severe ulcer score of grade 3 to 4). Lower levels of RBC GLA, DGLA, AA, and EPA correlated with severe squamous gastric ulceration (grade 3 to 4). CLINICAL RELEVANCE Equine gastric ulcer syndrome is prevalent in high-performance horses and is a concern to owners and trainers. Long-chain PUFA supplementation increased levels of GLA, DGLA, AA, EPA, and DHA, unlike SC-PUFA supplementation, and was associated positively with prevention or resolution of severe squamous gastric ulceration. Further studies are needed to evaluate different management styles and exercise intensities.