Hypertrophy of the ligamentum flavum (LF) can reduce the diameter of the spinal canal posteriorly. Such stenosis may significantly compress the dural sac and nerve roots, resulting in symptoms, even without a bulging anulus fibrosus or herniated nucleus pulposus. We conducted an anatomical study to determine the influence of age and gender on the thickness of the LF at the lower lumbar levels using magnetic resonance imaging (MRI). The thickness of 1,280 ligaments was determined at the L4-L5 and L5-S1 levels from MRIs. We screened 320 patients (152 males [47.5%] and 168 females [52.5%]) between 21 and 82 years of age. There were no significant differences in LF thickness with respect to gender (P > 0.05). Age was not correlated with the thicknesses of the LF. The left LF at the L4-L5 and L5-S1 levels was significantly thicker than on the right side (P < 0.05). Furthermore, the LF thicknesses at L5-S1 bilaterally were significantly greater than on the corresponding sides at L4-L5 (P < 0.05). The LF is an important anatomical structure, which might cause low back or leg pain. Therefore, the thickness of the LF should be measured and evaluated carefully in the case of spinal stenosis.