Queen mating frequency is an important reproductive trait of the western honeybee Apis mellifera. Yet, it demands more attention when investigated under extreme or confined ecosystems. Queen mating frequency of the Yemeni Honeybee A. m. jemenetica was estimated under Saudi Arabia desert conditions, Riyadh (24°71′36″N, 46°67′53″E). Mating of queens took place after 8–13 days from emergence. Duration of mating flight ranged between 26 and 39 min. Subsequently, six microsatellite loci were used to genotype queen's progeny (n = 30 workers/queen). The average number of drone alleles using workers genotypes ranged between 5.83 ± 0.31 and 6.33 ± 1.09. However, effective paternal allele number was extremely low and ranged between 3.35 ± 0.34 and 3.60 ± 0.40. This relatively low mating frequency of the Yemeni honeybee, A. m. jemenetica, might have striking effect on the overall colony survival. Providentially, this relatively low mating frequency does not impact colonial heterozygosity, shown in this study (0.66 ± 0.07–70 ± 0.04), adversely. These results may affect hive survivability and entails distinctive management practices under such conditions.