Specimens of cultured zebrafish acquired from different fish farms in Brazil may show genetic variability and alteration in allele frequency due to genetic drift and selective pressure in a captive environment, resulting in the differentiation of productive and reproductive characteristics. The aim of this study was to evaluate the genetic variability and reproductive characteristics of 180 zebrafish specimens from six Brazilian fish farms. A deviation from the Hardy-Weinberg equilibrium was observed in all evaluated stocks. Differentiation among stocks was observed in the amount of genetic variability with respect to observed heterozygosity and the inbreeding coefficient (FIS). Genetic distance between stocks was determined through the Fst index, and the formation of four distinct groups was observed by plotting the dendrogram based on Nei’s genetic distance. Differences were observed among reproductive parameters, such as the average number of eggs per female and hatchability. This second parameter proved to be related to the level of inbreeding of the population, whereas this effect was not observed for spawning frequency. We conclude that zebrafish stocks from the 6 different Brazilian fish farms present significant genetic and phenotypic variability. The genetic structure affects fecundity and should be considered when carrying out work where reproductive rates are evaluated.