Brycon amazonicus is a species native to Brazil, with significant socioeconomic importance and immense potential for fish production. It is the second most cultivated species in the Amazon. The lack of specific molecular markers limits genetic research. This study aimed to identify species-specific microsatellite markers for B. amazonicus and analyze the genetic diversity of four fish farms: Nova Mutum, Instituto Nacional de Pesquisa da Amazônia, Jatuarana, and Nova Airão. In addition, the transferability of these markers to species of the same genus (B. orbignyanus, B. falcatus, and B. gouldingi) was evaluated. Seventeen primer pairs were developed using the enriched library method. Eight of these were used for the genetic analysis of B. amazonicus stocks. In total, 47 alleles were identified. The mean endogamy coefficient (FIS) was negative and significant for Nova Mutum stocks. However, the populations of B. amazonicus in Instituto Nacional de Pesquisa da Amazônia (0.179), Jatuarana (0.099), and Nova Airão were positive and nonsignificant. Analysis of molecular variance showed that most of the variation was observed within the populations evaluated (57%), and genetic differentiation (FST = 0.423) among the stocks was high. Bayesian analysis indicated that the best number of genetic clusters was K = 3. Transferability testing showed successful amplification (90%) of the primers by estimating the allele size between 144 and 294 base pairs (bp) and a total of 24 alleles for the related Brycon species. This indicates the high potential of microsatellites for the analysis of diversity and population genetic structure in both the genus Brycon and family Characidae.