This study aimed to assess the feasibility of using RFLP of PCR-amplified 16S rRNA gene (s) by using universal primers 27f/1492r and a combination of three restriction enzymes, AluI, CfoI, and TaqI, for a low-cost, rapid screen for a primarily differentiation of isolates of the complex of aerobic spore-forming bacteria commonly found in honey samples. The described method produced unique and distinguishable patterns to differentiate among 80 isolates belonging to 26 different species of Bacillus, Brevibacillus, Lysinibacillus, Rummeliibacillus, and Paenibacillus reported in honey and other apiarian sources.