[Objective] Purpose of this work was to specifically screen the Gram positive (G⁺) bacteria of Bacillus that could encode the aconitate isomerase (AI) enzyme, to enrich our understanding of the distribution of AI and to provide theoretical and material basis for further research. [Methods] Through heat pretreatment of soil sample, plate cultivation by using ACO solid medium containing trans-aconitic acid (TAA) as the sole carbon source and the 16S rDNA sequences homologous analysis, the Bacillus target strains that encode AI can be isolated. [Results] We totally isolated 22 bacterial strains that could utilize TAA carbon from the ACO plate, and 16 of which were successfully classified as:2 strains of Bacillus megaterium, 7 strains of Bacillus aryabhattai, one Bacillus pumilus strain, and 6 undetermined Bacillus sp. strains; besides, we found that the AI-coding genes of these 16 Bacillus isolates were different from the already cloned ones in DNA sequence. [Conclusion] The species diversity of Bacillus bacteria encoding AI enzyme is rich, indicating the encoding ability in more G⁺ bacteria and updating the old opinion that considered AI distribution mainly in G^- hosts. Our research provided available microbial resource for further studies on the identification of AI gene as well as its biological significance.