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To determine the gender of a three-toed Box turtle, examine the plastron (the underside of the shell); males typically have a concave plastron, while females have a flatter one to accommodate egg-laying. Additionally, males often have brighter colors and more pronounced markings, and their eyes may be more vibrant compared to the females'. Lastly, males usually have longer, thicker tails that extend beyond the edge of the shell, whereas females have shorter tails.

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