To compare the practicability and tissue sample quality between different intestinal biopsy techniques. Experimental, randomized ex vivo study. Small intestine of nine horses. Four different biopsy techniques were evaluated in the aboral jejunum and the ileum within 1 h after euthanasia. One segment was used as control (C), and the applied techniques included an 8 mm biopsy punch (BP), transverse wedge resection (TW), longitudinal wedge resection with transverse closure (LW) and a longitudinal sample using Eppendorfer biopsy forceps (EF). Defects were closed using a single-layer continuous Lembert pattern. Duration of the procedure, intestinal diameter, contamination, and bursting pressure were determined. The quality of the obtained tissue samples for histological assessment was evaluated using a semiquantitative score. The jejunal and ileal samples were analyzed separately. All biopsy procedures including defect closure were completed within 5 min, with shorter closure times for BP (p = .03). Minimal contamination could be noted in 1/8 TW and 2/8 LW cases, without significant differences between the groups. Longitudinal closure techniques (BP, EF) showed more constriction than transverse closures (TW, LW) (p < .05). Bursting pressure was >75 mmHg in all cases. Technique BP showed significantly lower biopsy quality scores (p = .009). The tested biopsy techniques could all be applied effectively within a reasonable time frame, yet the biopsy punch was associated with significant artifacts and risk of missing mucosa. The findings provide insights into the possible advantages and limitations of the different techniques and alert the surgeon to potential issues with the quality of the tissue sample.