This study compared the collagen fibril diameter distribution among six anatomical sites of the rat medial collateral ligament (MCL). Ultrathin MCL sections from 4 male Sprague-Dawley rats were examined electron microscopically. With an automated quantitation method, 41,638 fibrils were measured and compared among the periphery and core regions of the femoral, middle, and tibial portions of the MCL. Results demonstrated significant difference (p < .0033) in mean fibril diameter distribution among the six sites. The mass-averaged diameters of the core and peripheral fibrils were between 175.53 to 190.82 nm and 88.47 to 109.18 nm, respectively, with the peripheral fibrils more homogeneous in size. The fibrils occupied 36.7% to 57.1% of the cross-sectional area of the ligament. About 50% of the fibrils had an oblique factor of 0.8-1.0, implying that most fibrils were aligned longitudinally. This study has provided a detailed profile of the collagen fibril distributions in rat MCL.