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Outbreaks of blastomycosis, caused by the fungus Blastomyces dermatitidis, occur in areas of endemicity in the United States and Canada, but the geographic range of blastomycosis is expanding. Previous studies inferred the location of B. dermatitidis through epidemiologic data associated with outbreaks because culture of B. dermatitidis from the environment is often unsuccessful. In this study, we used a culture-independent, PCR-based method to identify B. dermatitidis DNA in environmental samples using the BAD1 promoter region. We tested 250 environmental samples collected in Minnesota, either associated with blastomycosis outbreaks or environmental samples collected from regions of high and low endemicity to determine the basal prevalence of B. dermatitidis in the environment. We identified a fifth BAD1 promoter haplotype of B. dermatitidis prevalent in Minnesota. Ecological niche analysis identified latitude, longitude, elevation, and site classification as environmental parameters associated with the presence of B. dermatitidis. Using this analysis, a random forest model predicted the presence of B. dermatitidis in basal environmental samples with 75% accuracy. These data support the use of culture-independent, PCR-based environmental sampling to track spread into new regions and to characterize the unknown B. dermatitidis environmental niche. [ABSTRACT FROM AUTHOR]