The South American freshwater fish Tambaqui (Colossoma macropomum) is the most popular native species raised in Brazil. In the last two decades, producers have been using tambaqui (Colossoma macropomum) and two other Serrasalmidae species - Pacu (Piaractus mesopotamicus) and Pirapitinga (Piaractus brachypomus) - in terminal crosses to obtain hybrids resistant to lower temperatures and diseases, and with higher growth rates, replacing the parental species production in some regions in Brazil. Production of Tambaqui and its hybrids accounts for almost 30% of all finfish cultured domestically. Resulting interspecific hybrids are phenotypically indistinguishable from pure Tambaqui at juvenile stages and are fertile when sexually mature. Inadvertent crosses between pure species and fertile hybrids can negatively impact wild populations and captive purebred broodstocks. A database containing SNPs identified in a previous high-coverage genome-wide sequencing study with complexity reduction for these three species was analyzed to identify 55,295 candidate SNPs with species-specific alleles (Tambaqui≠Pacu=Pirapitinga). SNPType® assays were designed for 144 SNPs and used to test 140 samples used for SNP discovery (Tambaqui=63, Pacu=23, Caranha=48 and six known hybrids). Technical replicates were carried out to evaluate genotyping consistency. The best 96 markers were selected considering call rate and species-specific allelic concordance. Additionally, samples from six different commercial broodstocks (n= 288) of pure Tambaqui, Pirapitinga and known hybrids were tested with the 96-SNP panel, as a blind study. Genotyping results from a subset of 70 markers (after quality control pruning) were consistent with information received subsequently from producers, with the exception of three samples (one Tambaqui and two Pirapitinga), which showed a genotyping pattern consistent with undesired introgressions of up to 6%, corroborating the tool´s effectiveness to identify individuals with small introgressions. Power analysis estimates suggest that 54 independent markers are enough to detect a fourth backcross (3% contamination) with an error rate < 5%. This initiative represents initial step for broodstock certification in Tambaqui production system.