Vitellogenins (Vtg) are glycolipophosphoproteins synthesized by oviparous vertebrates as yolk proteins precursors. These proteins have been studied for their role in reproduction and in endocrine disruption. In this work, we report the first proteomic study towards the characterization of Vtg from Pseudoplatystoma fasciatum, Piaractus brachypomus and Colossoma macropomum. Male specimens of each of the three fish species were estradiol-induced (experimental) and non-induced (control). The initial Vtg characterization was made by 2D protein gel electrophoresis of both groups The identification of the high molecular weight spots, presumed to be Vtgs, was assessed by MALDI-TOF mass spectrometry analysis. A post-translational modification study was performed by differential staining of 2D gels in order to visualize phosphoproteins and glycoproteins. Plasma samples from the three species, induced with estrogen, showed three high molecular weight spots with variable isoelectric points. Post-translational modifications showed that Vtgs from P. brachypomus and C. macropomum presented a phosphorylated and glycosylated subunit, while the same subunit in P. fasciatum was only glycosylated. This characterization will help in the development of reliable immunoassays, which could be used for studies of endocrine disruption or for the improvement of artificial spawning, by uncovering the time of fish maturation or sex determination.