The Nile tilapia ( Oreochromis niloticus) plays a significant role in global aquaculture, with Brazil ranking among its primary producers. Nevertheless, a significant gap persists in the exploration of genetic diversity within Brazilian Nile tilapia stocks. To address this gap, we examined the genetic diversity of nine distinct farmed populations of Nile tilapia in Brazil, totaling 600 individuals, using single-nucleotide polymorphisms (SNPs). We sought to furnish essential genetic insights aimed at shaping the foundation of novel breeding nucleus. The pre-breeding populations exhibited high genetic diversity, with average heterozygosity ranging from 0.32 (SD 0.16) to 0.39 (SD 0.12). Some populations showed low effective population size (N _e) and higher probabilities of inbreeding (greater proportions of half- and full-sibling individuals). Despite the occurrence of cluster formation among populations, genetic differentiation between populations was minimal. Coefficients of relatedness among individuals were generated to facilitate mating strategies aimed at maintaining the genetic diversity of the populations. The presented results will ensure that effective strategies are implemented during the pre-breeding stage of a new tilapia genetic improvement nucleus, supporting the maintenance of genetic diversity in the breeding stock.