Coccidioides immitis is a fungal human pathogen endemic to semiarid soils in southern California and Baja California (Mexico). Results of culture-dependent detection of C. immitis in the past indicated a spotty distribution and unreliable prediction of C. immitis growth sites and accumulation sites. In this project we investigated bulk soil samples for the presence of the pathogen in nonagricultural loamy soils at nine different locations around Bakersfield, Kern County, California, for almost 2 y (2008-2009). To detect the pathogen we used a multiplex PCR method with optimized soil handling and storage, DNA extraction procedure and PCR protocol. With this method we were able to detect C. immitis in 8.42% of our samples in 2008 (n = 285), mostly from early spring to early summer. In 2009 however the percentage of samples positive for C. immitis from the same sites declined to 2.68% (n = 261). We also were able to distinguish C. immitis growth sites from accumulation sites. One site close to a recreation area (Lake Webb, Buena Vista Lake Basin), not previously known to support the growth of C. immitis, was identified as a strong growth site of the fungus. The cultivation-independent method in this study together with soil parameters can be used to predict and confirm C. immitis growth sites and might be a valuable tool for public health institutions.