Many relevant aspects of the breeding ecology of South American migratory fishes remain obscure. We conducted kinship analyses and parental reconstruction based on microsatellite genotypes of 41 Salminus brasiliensis larvae sampled from a single natural spawning event. Polygynandry involving 25 (12:13) potential parents was suggested, supporting ecological observations of aggregation during spawning. Some parents contributed up to five half-sibling larvae genotypes, implying a skewed contribution of genes to the progeny. This study contributed to preliminary insights into the S. brasiliensis genetic mating system.