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Feeding chickens diets high in n-3 fatty acids (FA) increases their incorporation into tissue lipids, but leads to oxidative stress in cells. This study investigated the effect of the dietary polyunsaturated FA ratio (PUFA n-6: n-3) and vitamin E (vE) level on DNA damage and morphological changes in the gut epithelium of chickens. One-day-old female broiler chicks (n = 176) were divided into 4 groups fed for 43 d diets with a high (HR) or low (LR) PUFA n-6: n-3 ratio and supplemented with 50 or 300 mg vE kg−1. Performance was calculated for periods of d 1 to 9, d 9 to 16, d 9 to 35, and d 9 to 42, while organs were sampled at d 9, d 17, d 36, and d 43. At d 17 and d 43, DNA damage of epithelial cells in the duodenum and jejunum was measured and duodenal and jejunal morphology was analyzed. HR diets improved FCR for the periods of d 1 to 9, d 9 to 16 and d 9 to 42, whereas the increased vE level improved FCR for the period of d 9 to 16. In the jejunum DNA damage was greater in chickens fed LR than HR diets at d 17 (P < 0.001) and the increased vE level promoted DNA damage in both intestinal segments (P < 0.02) in younger birds. The morphology of the duodenum was marginally affected by the diets, whereas LR diets in the jejunum reduced villus surface area at d 17 (P = 0.022), and mucosa thickness (P = 0.029) and villus height (P = 0.035) at d 43. The results indicated that feeding birds LR diets and vE levels significantly exceeding the recommendation induced DNA damage in epithelial cells, but this effect varied depending on the intestinal segment and the age of birds.