One of the main issues in the processing sector of the tambaqui Colossoma macropomum is the removal and/or fragmentation of intermuscular bones (IBs), which negatively impacts its production chain. In this sense, we quantitatively examined the IB variation in farmed tambaqui (n = 127) by comparing the direct anatomical dissection with the high-resolution X-ray imaging method. The number of IBs from the anatomical dissection on the left side of the fish (27.3 ± 5.70 bones) was comparable to that of X-ray analysis (26.9 ± 6.03 bones) (p > 0.05). In addition, 76% of deviation in IB number between the two studied methods was one to three, indicating both methods are equally efficient for identifying and quantifying IBs. We found a strong positive correlation (R = 0.8, p < 0.001) between the X-ray and the dissection methods. Our predictive models indicated that more than 50% of variation in IB length can be explained by growth parameters. Our results demonstrated that the X-ray method can provide accurate phenotypic data (in vivo) for IB counting and length measurements by extrapolating from the standard length, body weight and trunk over axis area of tambaqui.