OBJECTIVE: To develop a magnetic resonance imaging (MRI)-based approach for quantifying absolute fat mass in organs, muscles, and adipose tissues, and to validate its accuracy against reference chemical analysis (CA). METHODS: Chemical-shift imaging can accurately decompose water and fat signals from the acquired MRI data. A proton density fat fraction (PDFF) can be computed from the separated images, and reflects the relative fat content on a voxel-by-voxel basis. The PDFF is mathematically closely related to the fat mass fraction and can be converted to absolute fat mass in grams by multiplying by the voxel volume and the mass density of fat. In this validation study, 97 freshly excised and unique samples from four pigs, comprising of organs, muscles, and adipose and lean tissues were imaged by MRI and then analyzed independently by CA. Linear regression was used to assess correlation, agreement, and measurement differences between MRI and CA. RESULTS: Considering all 97 samples, a strong correlation and agreement was obtained between MRI and CA-derived fat mass (slope = 1.01, intercept = 1.99g, r(2) = 0.98, p < 0.01). The mean difference d between MRI and CA was 2.17±3.40g. MRI did not exhibit any tendency to under or overestimate CA (p > 0.05). When considering samples from each pig separately, the results were (slope = 1.05, intercept = 1.11g, r(2) = 0.98, d = 2.66±4.36g), (slope = 0.99, intercept = 2.33g, r(2) = 0.99, d = 1.88±2.68g), (slope = 1.07, intercept = 1.52g, r(2) = 0.96, d = 2.73±2.50g), and (slope=0.92, intercept=2.84g, r(2) = 0.97, d = 1.18±3.90g), respectively. CONCLUSION: Chemical-shift MRI and PDFF provides an accurate means of determining absolute fat mass in organs, muscles, and adipose and lean tissues.