BACKGROUND: Bone marrow adiposity and R2* have been explored as an imaging biomarker for osteoporosis. Chemical shift-encoded MRI (CSE-MRI) is a method that allows for relatively accurate measurement of adiposity and R2* in bone marrow in a simple manner. Additionally, there are reports of a physiological gradient of fat distribution in the lumbar spine. This physiological gradient of fat distribution can potentially impact the prediction of osteoporosis. Furthermore, the distribution of R2* is not well understood. PURPOSE: This study examined how lumbar spine fat fraction (FF) and R2* change with different levels of the lumbar spine, how they influence osteoporosis prediction, and how they change according to measurement methods. STUDY DESIGN/SETTING: Cross-sectional study using retrospectively collected data. PATIENT SAMPLE: The study included patients who underwent dual-energy X-ray absorptiometry and lumbar spine CSE-MRI within one-month intervals between 2017 and 2022. OUTCOME MEASURES: Reproducibility of FF and R2* based on measurement techniques, changes in FF and R2* according to vertebral level and osteoporosis status, and diagnostic power of osteoporosis based on vertebral level. METHODS: Patients were categorized into the normal bone density, osteopenia, and osteoporosis groups based on bone mineral density. The relationship between groups and spine level before and after BMD adjustment was investigated using generalized estimating equations. Comparisons between the three groups and various measures of reliability were conducted using intraclass correlation coefficient. The diagnostic performance for predicting osteoporosis was evaluated with a receiver operating characteristic curve. RESULTS: Comparing the three groups, FF increased with osteoporosis severity, while R2* decreased (p<.001). The intra/inter-rater agreement for FF and R2* was excellent. A physiological gradient within individuals was observed, where FF increased towards the lower lumbar spine (p=.002). R2* tended to decrease, but it was not statistically significant (p=.218). There was no statistically significant difference in the diagnosis of osteoporosis based on FF or R2* across different lumbar spine levels. CONCLUSIONS: There was an increase in FF and a decrease in R2* from T12 to L5. However, the predictive power of osteoporosis did not significantly differ between each level.