Honeybees play a vital role in the world’s food supply by acting as essential pollinators in the agricultural fields. Interestingly, more than one third of the world’s essential crops are honeybee’s dependant. The adult honeybeeworkers harbour a simple specific bacterial spectrum in their guts with vital role in bees’ health. Gut microbial diversity of adult honeybee workerswasstudied through targeting the V3 and V4 regions of the 16S rRNA geneviaIllumina MiSeq. The study identified four phyla of the gut microbiomesinadult workersof the two-honeybee subspecies A.m. jemeniticaandA.m. carnica. The most abundant phylum in microbiome of A.m. jemeniticawasFirmicutes (48%), while Protobacteria and Actinobacteriaphylawere less abundantat figures of31% and 10%, respectively. In microbiome of A.m. carnica,Firmicutes (57%) was also the most dominant phylum, while Protobacteria and Actinobacteria had lower prevalence at figures of 31% and 10%, respectively. At genus level, adult honeybee workers harboured a number ofLactobacillus spp.in their guts with relative abundance of 80% in A.m. jemeniticaworkers compared to52%forA.m. carnicaworkers.Up toour knowledge, this is the first study of its kind on gut microbiome diversity inhoneybee workersof different origins conducted in Saudi Arabia using high-throughput 16S rRNA gene sequencing technology. The results indicatedthat the variability inmonophyletic origin of host of honeybee workers affectedgut microbiota composition.