Brycon henni is a native species in Magdalena's River basin, and because of its cultural and economic importance, is strongly overexploited. This study aimed to describe the genetic variability and population structure of this species from Nare and Guatapé rivers basins. A total of 195 individuals were collected and DNA extractions were obtained from muscle and blood tissue. Fourteen primers were evaluated with the RAPD technique, being four of them polymorphic, and produced 66 different fragments (63% polymorphism). Besides, using the molecular variance (AMOVA) analysis, the population structure was described for all sites (phi(ST) = 0.297, phi(ST) = 0.163; Nare and Guatapé river basins, respectively), and suggested the importance of the migratory behaviour of the species in the genetic differentiation. Genetic distances among sampled sites showed that most of the genetic differentiation occurred between sites Guatapé and El Cardal with respect to the others. A Mantel Test demonstrated a correlation between genetic and geographic distances (r = 0.431 both of the basins evaluated; r = 0.377, Nare river middle basin), and suggested isolation by distance. The outcomes obtained in this study have valuable implications in species conservation and the genetic variability of natural populations of B. henni, and should be complemented with morphological analyses.