Knowledge about reproductive biology is critical for understanding species ecology and evolution, hence their conservation. However, such information remains elusive for many species due to the difficulty of observing mating in nature. Herein, we coupled population and sociogenetic analysis to assess the reproductive biology of an important South American catfish, the “painted catfish” Pimelodus maculatus. We analysed the genetic diversity and kinship patterns of three wild ichthyoplankton samples identified by DNA barcoding and screened with microsatellite loci. We found the same trend in the three samples analysed: a high number unrelated larvae (r < 0.25) and potential parents involved (Ca1, 24♀:24♂; Ca2, 21♀:23♂; Cin, 24♀:25♂), as well as a high number of familial clusters (ML: 19‐21; MCMC: 23‐26). The data indicate a parental sexual proportion close to 1:1 and a polygynandrous mating system. We found high levels of genetic diversity, low GST values (GST < 0.014), but moderate DEST values between the tributary and reservoir samples (DEST > 0.25; Cin x Ca1 or Cin x Ca2), a similar pattern to the observed in adult populations. These results indicate that the mating system of P. maculatus has played an important role in minimising the loss of genetic diversity, a condition that may have favoured the species in a highly fragmented landscape.