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Fungi play fundamental ecological roles in terrestrial ecosystems. However, their distribution and diversity remain poorly described in natural communities, particularly in arid and semi-arid ecosystems. In order to identify environmental factors determining fungal community structure in these systems, we assessed their diversity in conjunction with soil physicochemical characteristics in a semi-arid ecosystem in Baja California, Mexico, endemic for Coccidioidomycosis (Valley Fever). Two different microhabitats, burrows (influenced by rodent activity) and topsoil, were compared in winter and summer. Using a metagenomic approach, the ITS1 region of nuclear ribosomal DNA was used as barcode. A total of 1940 Operational Taxonomic Units (OTUs) were identified from 362 332 ITS1 sequences obtained by 454 pyrosequencing. Differences in fungal composition between seasons were clearly identified. Moreover, differences in composition between microhabitats were mainly correlated to significant differences in environmental factors, such as moisture and clay content in topsoil samples, and temperature and electrical conductivity in burrow samples. Overall, the fungal community structure (dominated by Ascomycota and Basidiomycota) was less variable between seasons in burrow than in topsoil samples. Coccidioides spp. went undetected by pyrosequencing. However, a nested PCR approach revealed its higher prevalence in burrows.