Management of invasive mold infections in patients with prolonged neutropenia and hematopoietic stem cell transplant (HSCT) recipients with graft-versus-host disease (GVHD) has been hampered by the difficulty in diagnosing these infections. Definite diagnosis invariably centers on histologic identification of hyphae in tissue or on culture from a sterile body site. Therefore, most practitioners have relied on prophylaxis and empiric therapy. Currently, emphasis is shifting from routine prophylaxis and empiric therapy to screening of patients with neutropenia at high risk so that clinicians can administer appropriate antifungal therapy early, when it can potentially improve patient outcome. Non–culture-based microbiologic tools are at the forefront of this paradigm shift. Commercially available methods to detect fungal antigens and sophisticated techniques to detect fungal DNA may be used as screening tools during the highest risk period. Together with assessment of clinical signs, cultures, and especially CT scanning, these methods are useful for starting antifungal therapy preemptively. While awaiting further evaluation of these tools during the postengraftment period of allogeneic HSCT, mold-active prophylaxis targeting the subgroup of patients with severe acute or chronic GVHD may be justified. However, some critical issues have not yet been adequately addressed, including the generalizability of study results, impact of mucositis and gastrointestinal GVHD on drug bioavailability, need for therapeutic drug monitoring, impact of prophylaxis on the performance of diagnostic assays, and optimal treatment of breakthrough invasive fungal infections.