Gunshot residue (GSR) evidence may be altered or obscured by after-death events such as putrefaction, autolysis, and/or damage by animals. The present study aimed at evaluating and comparing the amount and differential distribution of GSR utilizing microcomputed tomography (microCT) analysis of fresh and decomposed gunshot wounds. A total of 60 experimental shootings at three different firing distances (5, 15, and 30 cm) were performed on human calves surgically amputated for medical reasons. Thirty specimens (10 for each tested distance) were immediately formalin-fixed, while the other 30 specimens were enclosed in a cowshed for 15 days, before formalin fixation (air temperature ranging from 11°C to 38°C). MicroCT analysis with three-dimensional image reconstruction detected GSR particles in all the investigated entrance wounds. In fresh specimens, GSR was concentrated on the skin surface around the entrance hole and in the epidermis and dermis layers around the cavity, while in decomposed specimens, the high density particles were detected only in the dermis layer. No GSR was detected in exit wounds of both fresh and decomposed specimens regardless of the tested firing distance. Statistical analysis demonstrated that also in decomposed wounds the amount of GSR roughly correlated with the distance from which the gun was fired, exhibiting, however, a higher variability than in fresh samples. The obtained results suggest that microCT analysis can be a valid screening tool for differentiating decomposed entrance and exit gunshot wounds.