This study aimed to evaluate and compare the proximate composition and fatty acids (FA) profile of four fish species Piaractus mesopotamicus (PM), Colossoma macropomum (CM), Piaractus mesopotamicus X Colossoma macropomum (PC) and Piaractus brachypomus (PB). Fish fillets were analyzed for proximate composition, fatty acid profile, and nutritional quality indices. In general, PM and PB presented lower moisture content (73.75 ± 0.07 and 70.47 ± 0.16) and higher lipid content (4.34 ± 0.81 and 9.23 ± 1.16), respectively (p < 0.05). Although all species presented the same PUFA>MUFA>SFA pattern, PM and PB showed higher total n-3 FA values (mainly DHA and EPA), while CM had the lowest one (p < 0.05). Moreover, PM and PB exhibited superior nutritional quality indices, presenting the highest h/H ratio and the lowest IA and IT ratio (p < 0.05). A similar nutritional profile was observed for CM and PC. Therefore, PM and PB represent an excellent source of high-quality lipids, demonstrating that these freshwater fish species can be considered optimal for human consumption.