The presence of intermuscular bones in fisheries products limits the consumption and commercialization potential of many fish species, including tambaqui (Colossoma macropomum). These bones have caused medical emergencies and are an undesirable characteristic for fish farming because their removal is labor-intensive during fish processing. Despite the difficulty in identifying genes related to the lack of intermuscular bone in diverse species of fish, the discovery of individuals lacking intermuscular bones in a Neotropical freshwater characiform fish has provided a unique opportunity to delve into the genetic mechanisms underlying the pathways of intermuscular bone formation. In this study, we carried out a GWAS among boneless and wt tambaqui populations to identify markers associated with a lack of intermuscular bone. After analyzing 11 416 SNPs in 360 individuals (12 boneless and 348 bony), we report 675 significant (P-adj < 0.003) associations for this trait. Of those, 13 associations were located near candidate genes related to the reduction of bone mass, promotion of bone formation, inhibition of bone resorption, central control of bone remodeling, bone mineralization and other related functions. To the best of our knowledge, for the first time, we have successfully identified genes related to a lack of intermuscular bones using GWAS in a non-model species.