In fish, the fat content contributes to promoting the nutritional and organoleptic characteristics of the flesh, which is crucial for consumer acceptance. Methods to predict the fat in fish are important in nutritional and physiological research, where body content is traditionally determined by dissection followed by chemical analysis. However, X-ray micro-computed tomography (micro-CT) provides three-dimensional information in a non-destructive way. This work aims to characterize radiologically the fat, in situ, in a widely cultivated marine species, gilthead seabream (Sparus aurata). To validate the method changes in fat content in a control group (fed) and another group (unfed for 60 days) were assessed. Fish images were acquired on an Albira SPECT/PET/CT preclinical-scanner. Image analysis and measurements were performed using the Carestream Molecular Imaging Albira CT system in conjunction with Pmod and Amide packages. By micro-CT analysis the density values were determined for the whole fish body (- 1,000 to + 2,500 HU, Hounsfield units), and density ranges for the fat in S. aurata were established from - 115 to + 50 HU. As expected, significant differences were found between fed and starved groups at 60 days. The present study confirms the usefulness of high-resolution morphological analysis for evaluating the presence and distribution of fat in this important fish species.