The farming of Colossoma macropomum has intensified in recent years, leading to an increased need for research into the health of the fish. We therefore investigated the diversity of myxosporeans (Cnidaria: Myxozoa) infecting C. macropomum in a breeding system in the municipality of Rio Branco, in the state of Acre, Brazil. Twenty-four fish specimens were examined from June to August 2018. Our results revealed a high prevalence of infection, with 23 specimens (95.8%) exhibiting myxosporean plasmodia. Morphological analysis, based on light and electron microscopies, and molecular analysis (small subunit ribosomal DNA [SSU rDNA] sequencing) revealed the occurrence of three novel species of the genus Myxobolus. Plasmodia of Myxobolus guttae n. sp. were found in the fins of 75% of the specimens, and the myxospores were pear-shaped, measuring 12.3 ± 0.6 (10.3–13.5) μm in length, 8.1 ± 0.3 (7.1–8.6) μm in width, and 5.1 ± 0.6 (4.5–6.5) μm in thickness. The polar capsules were elongated and equal in size, measuring 6.8 ± 0.5 (5.8–7.6) μm in length and 2.5 ± 0.3 (1.8–3.1) μm in width, exhibiting polar tubules with 9–10 coils. The plasmodia of Myxobolus longus n. sp. were found in the gills of 45.8% of the C. macropomum specimens, and the myxospores were fusiform, measuring 16.4 ± 0.6 (14.7–17.3) μm in length, 7.1 ± 0.2 (6.8–7.7) μm in width and 5.5 ± 0.6 (4.4–6.6) μm in thickness. The polar capsules occupied more than half of the myxospore, exhibiting different sizes, the largest measuring 9.2 ± 0.5 (7.3–10.1) μm in length and 2.5 ± 0.3 (2.0–3.1) μm in width, while the smallest measured 8.5 ± 0.4 (7.1–9.1) μm in length and 2.4 ± 0.2 (1.9–3.0) μm in width. Both polar capsules contained polar tubules with 10–12 coils. For Myxobolus tambaquiensis n. sp., plasmodia were found in the opercular cavity of 41.7% of the fish specimens, and the myxospores had an oval shape, measuring 10.0 ± 0.4 (9.5–11.3) μm in length, 6.5 ± 0.2 (6.1–7.1) μm in width, and 4.6 ± 0.4 (3.9–5.6) μm in thickness. The polar capsules were elongated and equal in size, measuring 4.9 ± 0.2 (4.4–5.3) μm in length and 1.9 ± 0.2 (1.5–2.2) μm in width, closing with 8–9 coils of the polar tubule. The morphological and sequencing data of the SSU rDNA showed that the three species studied herein remain unknown to science, increasing the diversity of myxosporeans infecting C. macropomum, an iconic fish in South American freshwater fish farming. The SSU rDNA based phylogenetic analysis revealed that Myxobolus spp. parasites of C. macropomum did not have a monophyletic origin, identifying different times and pathways of the acquisition of parasites by this host species.