[Objectives] The Bider marking is a distinctive feature on horses, characterized by unique and symmetrical black irregular patterns located on the shoulder blades. Currently, this marking is found exclusively on Przewalski’s Horse Equus ferus przewalskii and Mongolian Horses E. caballus that exhibiting a Dun coat color. This study aims to explore the differences in the formation of skin and hair follicles between the Bider marking area (shoulder), the dorsal midline and croup areas of Dun Mongolian Horses. [Methods] Paraffin sectioning and hematoxylin and eosin (H.E) staining techniques were employed to observe and analyze the morphological structure of hair follicles, patterns of pigment deposition, epidermal thickness, and hair follicle density in the skin regions of the shoulder, dorsal midline, and croup of Mongolian Horses. Skin tissue samples were collected from the shoulder, dorsal midlines, and croup of six Dun Mongolian Horses with Bider markings (Fig. 1). These samples underwent paraffin embedding, sectioning (both transverse and longitudinal), H.E staining, and subsequent microscopic observation using a BX53 (LED) semi-electric fluorescence microscope. To assess the symmetry of the anagen hair bulbs, five unique microscopic images of anagen hair bulbs from the shoulder, dorsal midline, and croup were selected through longitudinal sectioning. The hair bulbs were divided into left and right sides based on the central line of the hair papilla. t-test was performed on data exhibiting uniform hair follicle morphology, with data values for the left and right sides of each hair bulb recorded separately. Hair follicle density and epidermal thickness were measured using both transverse and longitudinal sectioning preparation methods. Continuous sections containing hair follicle shafts and the epidermal layer were selected from skin tissues of the shoulder, dorsal midline, and croup, all observed under consistent microscopic conditions. Five representative images were selected for measurements. Hair follicle counting involved tallying the number of clearly visible hair follicles in each selected image, with the total count recorded for each field of view. To measure epidermal thickness, Image View image processing software was employed to obtain precise measurements within the epidermal layer of each image, and the corresponding values were documented. t-test was applied to determine whether significant differences existed in hair follicle density and epidermal thickness across different areas (shoulder, dorsal midline, croup). Statistical analysis of the data was conducted using GraphPad Prism 10 software. [Results] Following longitudinal sectioning and H.E staining of skin tissues (Fig. 2a), the epidermal layer (Fig. 2b), dermal layer, hair follicle structure, and sebaceous glands were examined. The hair follicle structure included the hair shaft, hair bulb (Fig. 2c), hair root, inner root sheath, outer root sheath, and hair papilla. Transverse sectioning facilitated the observation of the hair cortex, medulla, and cuticle structures of the hair shaft (Fig. 2d). Longitudinal sectioning of skin tissues from the shoulder, dorsal midline, and croup (Fig. 3) revealed the structural integrity of the skin tissues and hair follicles. The shoulder (Fig. 3a) and croup (Fig. 3c) were in the anagen phase, while the dorsal hair follicles exhibited atrophy (Fig. 3b), indicating they were in the catagen phase. Pigment deposition was observed in the epidermal layer and hair bulb regions of the dorsal midline, shoulder, and croup. Hematoxylin and eosin (H.E) staining revealed significant melanin deposition in the epidermal and hair bulb areas, facilitating the distinction between eumelanin and pheomelanin. Notably, significant asymmetry in pigment deposition was observed in the anagen hair bulbs of the croup. However, this asymmetry was not observed in the shoulder and dorsal midline (Fig. 4). In the comparative observation of pigment deposition in anagen hair bulb regions (Fig. 5), it was noted that the pigment distribution on the shoulder (P = 0.866) and dorsal midline (P = 0.093) exhibited symmetrical characteristics, with no statistically significant difference in pigment deposition area between the left and right sides. Conversely, pigment deposition on the croup displayed significant asymmetry (P = 0.049). In our comparison of follicle density among the shoulder, dorsal midline, and croup coats (Fig. 6a), we observed highly significant differences in follicle density between the croup and shoulder (P = 0.002) as well as between the dorsal midline and shoulder (P = 0.001). When examining the thickness of the epidermal layers in the shoulder, dorsal midline, and croup coats (Fig. 6b), we found that the difference in epidermal layer thickness between the dorsal midline and croup was not significant (P = 0.558). Additionally, the difference in epidermal layer thickness between the dorsal midline and shoulder was also not significant (P = 0.059). However, a significant difference in epidermal layer thickness was observed between the croup and shoulder (P = 0.020). [Conclusion] This study offered a comprehensive examination of the morphological characteristics of skin and hair follicles, along with pigment deposition patterns, in various coat regions of Dun Mongolian Horses with Bider markings. These findings provide an important foundation and reference point for future research on the morphological basis of the distinctive Bider marking in Mongolian Horses.