Studies on genetic composition in fish populations contribute to conservationist practices and inbreeding control in fish stocks. To this end, molecular tools such as microsatellite markers (SSRs) are often used, but they are expensive and time-consuming to develop. A species-specific heterologous marker emerges as an alternative, which can be used in taxonomically related species in a fast way. Our goal was to test SSRs markers of Brachyplatystoma rousseauxi and Pseudoplatystoma punctifer in P. reticulatum in an unprecedented way. For this purpose, DNA was extracted from fragments of the caudal fin of 222 P. reticulatum adults, using a NaCl-based method. Then, DNA samples were amplified by Polymerase Chain Reaction (PCR) using six markers, four from B. rousseauxi (BR38, 47, 51, and 61) and two from P. punctifer (PPU13 and PPU15). Two primers showed non-specific amplification and were disregarded (BR38 and PPU13). In the remaining four primers, the number of alleles per locus varied between two (BR47) to sixteen (BR51), and the average size of alleles was between 142 and 400 bp. Mean effective number of alleles per locus ranged from 10,650 (BR51) to 1,784 (BR47), with null or low-frequency alleles in all studied loci. Observed heterozygosity ranged from 0.299 (BR47) to 0.640 (BR51) and was always lower than the expected heterozygosity. Hardy-Weinberg balance was significant (p < 0.05) in all loci, and inbreeding coefficient (FIS) was always positive. Polymorphic Information Content (PIC) confirmed the efficiency of the markers since they had moderate (BR47) to high levels of information (BR51, BR61, and PPU15). Transferability test showed that the heterologous microsatellite molecular markers, originally for B. rousseauxi and P. punctifer, were efficient in P. reticulatum, producing three primers with high information content. Therefore, these markers can be safely used in future population studies of this species.
