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The detection and identification of bio-threat agents and the study of host-pathogen interactions require a high-resolution detection platform capable of discerning closely related species. Diverse analysis methods are used to identify pathogens, specifically Brucella species or biovars. In this study, we compared four diagnostic approaches including serology-based biochemical test, PCR assay, microarray analysis using a Universal Bio-signature Detection Array (UBDA) and whole genome “deep” sequencing for Brucella organisms including a number of field isolates. We found that although there was frequent agreement among the different tests, some tests gave compound/contradictory results that were a consequence of species diversity due to mixed infections or minor contaminants as measured by UBDA and validated from whole genome sequence. By comparing these analysis techniques, we demonstrate that standard diagnostics used in the field are limited in their ability to identify genomic DNA contaminants in field isolates while UBDA and sequencing analysis are highly sensitive in tracing genomic differences among the isolates.