Culture-independent molecular techniques such as real-time PCRs offer the potential for early diagnosis of invasive aspergillosis (IA), thereby reducing the disease-associated mortality rate. PCR-based testing is presently excluded from disease-defining consensus criteria due to lack of standardization and clinical validation. A single-center prospective study was conducted to investigate the performance of the commercially available MycAssay Aspergillus test for detecting Aspergillus DNA in patients with suspicion of IA. To this end, a total of 158 bronchoalveolar lavage (BAL) fluid specimens that were consecutively collected from hematology (n = 68) and intensive care unit (n = 90) patients were examined. Sixteen of 17 (94.1%) specimens from patients with proven/probable IA were MycAssay positive, and 15 of these 16 patients were also positive by an "in-house" PCR assay. A total of 139 of 141 (98.6%) specimens from patients without proven/probable IA were MycAssay negative. Fifteen of 16 (94.1%) MycAssay-positive patients were also positive for BAL fluid galactomannan (GM) at an index cutoff of ≥1.0 (index range, 1.1 to 8.3), as were 3 patients without IA but with pulmonary fusariosis. Interestingly, in seven of the PCR-positive BAL specimens that tested culture positive for Aspergillus species, cycle threshold values were earlier than those of specimens with a culture-negative result. In conclusion, the MycAssay Aspergillus PCR appears to be a sensitive and specific molecular test for the diagnosis of IA, and its performance is comparable to that of the GM assay. However, more large studies are necessary to firmly establish its clinical utility in high-risk settings.