Strategies for environmental pathogen monitoring must be specific for the target organism and sensitive across a wide range of environments. Air, water, and soil environments contain a complex, diverse assemblage of natural bacterial species that constitute the normal background upon which biothreat surveillance and forensic activities must operate. To provide a context for interpreting DNA-based detection assays from environmental samples, we have conducted surveys of aerosols and soils in many U.S. cities. Our surveys focused on the natural occurrence of 5 bacterial pathogens and their closely related species that can share considerable genomic and physiological traits. Our strategy employed PCR assays designed to detect (a) the pathogen target plus its close relatives, (b) virulence determinants and (c) housekeeping genes capable of differentiating bacterial subspecies, followed by sequencing and phylogenetic analysis. Using this approach, we determined that non-pathogenic species closely related to some of the pathogens are widespread in soils and aerosols. We also discovered new environmental species and subspecies related to target pathogens, for which we have no information on pathogenicity. Widespread distribution of such closely related organisms may have implications for virulence gene transfer, as well as for our ability to specifically detect biothreat agents in environmental samples.