New or emerging infectious diseases are commonly caused by pathogens that cannot be readily manipulated or studied under common laboratory conditions. These limitations hinder standard experimental approaches and our abilities to define the fundamental molecular mechanisms underlying pathogenesis. The advance of capped small RNA sequencing (csRNA-seq) now enables genome-wide mapping of actively initiated transcripts from genes and other regulatory transcribed start regions (TSRs) such as enhancers at a precise moment from total RNA. As RNA is nonpathogenic and can be readily isolated from inactivated infectious samples, csRNA-seq can detect acute changes in gene regulation within or in response to a pathogen with remarkable sensitivity under common laboratory conditions. Studying valley fever (coccidioidomycosis), an emerging endemic fungal infection that increasingly impacts livestock, pet, and human health, we show how csRNA-seq can unravel transcriptional programs driving pathogenesis. Performing csRNA-seq on RNA isolated from different stages of the valley fever pathogen Coccidioides immitis revealed alternative promoter usage, connected cis -regulatory domains, and a WOPR family transcription factor, which are known regulators of virulence in other fungi, as being critical for pathogenic growth. We further demonstrate that a C. immitis WOPR homologue, CIMG_02671, activates transcription in a WOPR motif-dependent manner. Collectively, these findings provide novel insights into valley fever pathogenesis and provide a proof of principle for csRNA-seq as a powerful means to determine the genes, regulatory mechanisms, and transcription factors that control the pathogenesis of highly infectious agents. IMPORTANCE Infectious pathogens like airborne viruses or fungal spores are difficult to study; they require high-containment facilities, special equipment, and expertise. As such, establishing approaches such as genome editing or other means to identify the factors and mechanisms underlying caused diseases, and, thus, promising drug targets, is costly and time-intensive. These obstacles particularly hinder the analysis of new, emerging, or rare infectious diseases. We recently developed a method termed capped small RNA sequencing (csRNA-seq) that enables capturing acute changes in active gene expression from total RNA. Prior to csRNA-seq, such an analysis was possible only by using living cells or nuclei, in which pathogens are highly infectious. The process of RNA purification, however, inactivates pathogens and thus enables the analysis of gene expression during disease progression under standard laboratory conditions. As a proof of principle, here, we use csRNA-seq to unravel the gene regulatory programs and factors likely critical for the pathogenesis of valley fever, an emerging endemic fungal infection that increasingly impacts livestock, pet, and human health.