Brazilian native meliponines are currently threatened by increased human impacts. The assessment of their genetic variation by microsatellite DNA markers can assist in the conservation of populations and help in the planning and establishment of efficient management strategies. The purpose of this study was to develop the first set of microsatellite markers for Melipona fasciculata, selected from partial genome assembly of Illumina paired-end reads. Primer pairs were designed for each detected locus at their flanking regions. Bee samples were genotyped from two different populations of Northeastern Brazil for marker characterization and validation. A total of 17 microsatellite loci displayed polymorphism. Mean HE and HO heterozygosities were 0.453 and 0.536, respectively. PIC across all loci ranged from 0.108 to 0.714. A genetic diversity analysis revealed high values for population differentiation estimates (FST = 0.194, RST = 0.230, and Dest = 0.162) within the investigated region. PCoA and Bayesian clustering showed a separation of the species into two distinct clusters. These microsatellite markers have demonstrated strong potential for population-level genetic studies. Moreover, the preliminary analysis of the genetic diversity in M. fasciculata provides provisional evidence of significant population differentiation between the two studied populations., Geice Ribeiro Da Silva, Isis Gomes De Brito Souza, Fabia De Mello Pereira, Bruno De Almeida Souza, Maria Teresa Do Rego Lopes, Paul Bentzen, Fabio Mendonça Diniz., and Obsahuje bibliografii