PCR-RFLP (Polymerase Chain Reaction-Restriction Fragment Length Polymorphism) analysis is used to detect genetic variability and population structure in mitochondrial DNA of Thai giant honeybees (A. dorsata) from 5 geographic areas (north, north-east, central, south and Samui Island). PCR amplification using specific primers flanking to intergenic COI-COII region, CytbI-tRNASer gene, ATPase6-8 gene and lrRNA gene yielded PCR products of 1040, 845, 820 and 750 bp, respectively. Digestion of these PCR products with Dra I and Hinf I revealed one, one, one and two haplotypes and three, four, and one haplotypes, respectively. Six composite haplotypes were generated from these study. Geographic heterogeneity in the distribution frequency was determined using c2 test. Six composite haplotypes did not show any significant differences in the distribution frequency in all pairwise comparisons. Therefore, determination of mtDNA by PCR-RFLP did not a sensitive marker for studying intraspecific variation A. dorsata in Thailand.Three microsatellite loci (A14, A24 and A88) were used to study on population genetic of A. dorsata in 5 geographic areas by 13 A. mellifera microsatellite loci. These microsatellite were shown to be polymorphic with number of alleles at each locus of 6, 8 and 20 alleles, respectively. Population analyses based on 3 loci revealed heterozygosity between 0.68-0.74 and average number of alleles per locus were ranged 6.0-9.0. The analysis of geographic heterogeneity, the A24 locus indicated that the allele distribution frequencies of A. dorsata in Samui Island was significant difference from mainland (north, north-east, central and south). Microsatellite locus A24 can be used to investigate genetic differentiation of A. dorsata from mainland and Samui Island of Thailand.