Aim. To determine the level of the conus medullaris-Tuffier's line, and conus medullaris-Tuffier's line distance using imaging and evaluate their relation to age and gender. Methods. We performed a cross-sectional study of 189 adult participants, who underwent MR imaging of lumbosacral spine. Each vertebra was divided into 3 equal segments (upper, middle, and lower), and intervertebral disc space was also assumed as one segment. All segments from T12 upper segment to L5S1 intervertebral disc were numbered consecutively. The position of conus medullaris and Tuffier's line was determined by the vertebral segment or intervertebral disc space at the same level. The patients were stratified into high/low conus medullaris position (cutpoint: L1 middle segment) and short/long conus-Tuffier's distance (cutpoint: 14 segments). Results. Women with low conus were significantly more than men, in patients older than 50 years old (72.7% in females versus 55.3% in males; P < .05), whereas there was not such a sexual dimorphism in patients younger than 50 years old. Similarly, short conus-Tuffier's distance was more frequent among women than men in patients older than 50 years old (59.7% in females versus 39.5% in males; P < .05), whereas there was not any gender difference in patients younger than 50 years old. Conus-Tuffier's distance was negatively correlated with age (r = −0.32, P < .001) in all studied population. Conclusion. Anatomical landmarks vary according to age and gender, with a lower end of conus medullaris in women, so clinicians should use more caution on the identification of the appropriate site for lumbar puncture, particularly in elderly women.