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A systematic study of regional skin thickness in different types of horses has not yet been described. Knowledge about regional skin thickness has long been used in human medicine to optimise wound care and skin grafting outcomes and similar knowledge could prove useful in equine wound management. The objective of the current study was to report the topographical variation in skin thickness of Warmblood (WB) and Coldblood (CB) type horses and to compare different methods for measuring skin thickness. Horses free of skin conditions and euthanised for reasons unrelated to this study were included. Skin biopsies were collected post-mortem from 28 locations in 9 WB horses and skin biopsy thickness was measured using a digital calliper. In 6/28 locations, skin fold measurements using a micrometer were also obtained. In another cohort comprising 8 WB and 10 CB horses, skin biopsies were harvested from 6 locations for histologic skin thickness measurements. Descriptive statistics revealed large topographical variation in skin thickness. A mixed effect model assessing the effect of breed and sampling location demonstrated that skin thickness was significantly higher in CBs than WBs (P < 0.001). At the mid-15th rib and between the forelimbs, there was strong correlation between calliper and skin fold measurements (ρ 0.72 and 0.74, respectively), whereas correlation was very strong at the ventral abdomen (ρ 0.83). In conclusion, this study demonstrates large topographical variations in skin thickness in horses, and significant differences between horse types. Skin fold measurements may estimate skin thickness at the ventral abdomen.