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Abstract In the South‐West Indian Ocean, the honeybee Apis mellifera is found on several islands including the Seychelles archipelago. This archipelago is located 1120 km North of Madagascar, where the endemic African subspecies A. m. unicolor occurs. The genetic diversity of the honeybee populations in the Seychelles islands has never been studied, yet this species interacts with highly endemic and indigenous flora. A total of 186 honeybee colonies from the three main islands: Mahé, Praslin and La Digue were collected. In addition, 107 individuals from Madagascar ( A. m. unicolor ) and 49 from Italy ( A. m. ligustica ) were analysed as reference populations. The maternal lineages were assessed using PCR ‐ RFLP ( n = 342) and sequencing ( n = 121) of the mt DNA COI – COII intergenic region. Intra‐Seychelles nuclear genetic diversity and structure were analysed using 15 microsatellites while comparison with reference populations was done using 14 loci. All Seychellian colonies had mt DNA sequences characteristic of the African evolutionary lineage. Two sub‐lineages were detected: A I sub‐lineage (A1) was dominant (96.7%) on all islands and mostly represented by the subspecies A. m. unicolor, while Z sub‐lineage was observed in six colonies from two islands. No mt DNA characteristic of imported European lineages was detected. Nuclear genetic diversity was high and structured, suggesting restricted gene flow between islands of the archipelago. High nuclear similarities were found among the Seychellian and A. m. unicolor populations, yet significant genetic differentiation was observed. The A. m. ligustica reference population was highly differentiated from the Seychellian honeybee populations.