Apis mellifera unicolor is a tropical honeybee endemic of Madagascar. Comprehensive knowledge about its mitochondrial and nuclear genetic diversity and structuration was our main purpose. Samples of worker bees were collected from 867 colonies in 76 sites in Madagascar and 1 reference population in South Africa. PCR-restriction fragment length polymorphism (RFLP) and sequencing were used to reveal variability in the COI–COII mtDNA region. Seventeen microsatellite loci were used for studying the nuclear diversity. Three PCR-RFLP profiles were observed, among which 99.4 % belonged to A1 haplotype, 0.2 % to a new A haplotype, and 0.4 % to A4 haplotype. In microsatellite analysis, moderate genetic diversity values were found for Madagascar, together with low mean number of alleles ranging from 2.47 to 3.88 compared to South Africa. Bayesian clustering assignment methods and principal component analysis (PCA) separated populations into two genetic clusters which matched with geographic areas. Several hypotheses are discussed regarding to the low genetic diversity of A. m. unicolor in its native range.