Purpose: To determine differences in fat-signal fraction (FF) from chemical-shift-encoded water-fat MRI of interscapular BAT in mice housed at different ambient temperatures (Ta). Materials and Methods: C57BL/6J male mice (8 weeks old) were singly housed at 16°C, 23°C, or 30°C (n = 16/group) for 4 weeks. Measures included food intake, body weight (both measured weekly) and body composition (at baseline, 2, and 4 weeks post-thermal exposure); chemical-shift-encoded water-fat MRI was performed on a 9.4 Tesla Bruker magnet with respiratory gating and anesthesia at 4 weeks post-thermal exposure. Results: A significant inverse relationship between food intake and Ta was evidenced (P < 0.0001). Lean mass was similar among groups, while total fat mass was significantly different among groups ([mean ± SE]: 30°C = 5.10 ± 0.19 g; 23°C = 4.18 ± 0.16 g; 16°C = 3.48 ± 0.54 g; P < 0.0001). Mean BAT-FF was positively related to Ta (means: 30°C = 79.4%; 23°C = 61.8%; 16°C = 50.9%; P < 0.0001). Conclusion: These cross-sectional results demonstrate that MRI measurement of FF within the interscapular BAT in mice reflects recent functional status of the tissue, with a lower Ta leading to a significantly reduced BAT-FF, indicative of the tissue's involvement in thermogenesis. © 2013 Wiley Periodicals, Inc.