Natural populations of tambaqui (Colossoma macropomum) have significantly decreased in recent decades especially due to human extraction activities. So that the environmental impact may be reduced, the restocking of fish and increase in fish production are enhanced. Genetic evaluations using molecular markers are essential for this purpose. Current study evaluates the genetic variability of two tambaqui broodstocks used in restocking programs. Sixty-five samples (33 samples from broodstock A and 32 samples from broodstock B) were collected. DNA was extracted from caudal fin samples, with the amplification of four microsatellite loci: Cm1A11 (EU685307) Cm1C8 (EU685308) Cm1F4 (EU685311) and Cm1H8 (EU685315). Fourteen alleles in the stock of broodstock A were produced, five alleles for Cm1A11 locus (230, 255, 260, 270 and 276 bp), three alleles Cm1C8 (239, 260, and 273 bp), two alleles Cm1F4 (211 and 245 bp), four alleles for Cm1H8 (275, 290, 320 and 331 bp) and two unique alleles were found for Cm1A11 loci (alleles 270 and 276 bp) and Cm1H8 (alleles 275 and 331 bp). In broodstock B, ten alleles were produced, the same alleles of the first stock except for alleles 270 and 276 bp in Cm1A11 locus and 275 and 331 bp in Cm1H8 locus. Broodstock A revealed low frequency alleles in Cm1A11 loci, Cm1C8, Cm1F4 and Cm1H8, whereas broodstock B had no locus with low allelic frequency. Loci Cm1A11, Cm1C8 and Cm1H8 exhibited significant deficit of heterozygotes in both broodstocks, revealing changes in Hardy-Weinberg equilibrium. Genetic diversity between stocks was 0.1120, whilst genetic similarity was 0.894, with FST rate = 0.05, and Nm = 3.93, indicating gene flow between the two broodstocks. Results show that broodstocks are genetically closely related, with no great genetic variability. Strategies such as a previous genetic analysis of breeding with its marking, use of a large Ne crossing between the most genetically divergent specimens, and the introduction of new genetic material to broodstocks may maximize genetic diversity and minimize inbreeding within the next generation.