Burkholderia multivorans ATCC 17616 was originally isolated from a soil sample, and it carries three chromosomes. To identify traits of likely adaptive significance for colonization of soil, an in vivo expression technology system for ATCC 17616 was constructed using the promoterless and tandemly arranged dapB and lacZ genes as the reporters, and this system was applied to identify the genomic loci of ATCC 17616 that were induced in sterilized soil. Our screening of a library consisting of dapB-lacZ-inserted clones resulted in the isolation of 713 clones in which the insertion sites of genome were putatively transcribed in the soil but not in laboratory media. All insertion sites in the genome were determined by high-throughput sequencing using genomic DNA as the templates, and subsequent analysis led to a reliable list of a total of 116 genomic loci as the B. multivorans ATCC 17616 loci induced in a soil environment (mls). These 116 mls carried the genes for energy acquisition from various substances, as well as genes for cell-envelope integrity and the niche adaptation. The distribution of these loci was biased to the second chromosome, suggesting the importance of this replicon as a source of adaptive traits enhancing survival of this organism in natural environments.