Diagnosis of invasive aspergillosis remains a significant problem. PCR testing may aid diagnosis but is not yet included in disease-defining criteria due to a lack of standardization of assays and methodologies. This study investigated the analytical performance and the clinical sensitivity and specificity of the Myconostica MycAssay Aspergillus PCR (MAP) assay compared to those of a validated in-house Aspergillus PCR (IHP) test when testing serum specimens. Serum specimens spiked with Aspergillus genomic DNA had a limit of detection equivalent to 5 genomes and a linear dynamic range of 5 to >5 × 10(4) genomes for both assays. When testing clinical specimens (n = 170), the MAP assay had a sensitivity of 60 to 70% and a specificity of 90.5 to 100%. The IHP assay had a sensitivity of 50 to 80% and a specificity of 100%. A commercially available Aspergillus PCR assay provides a methodology that is standardized and reagents that are quality controlled. This facilitates multicenter evaluation of the clinical utility of PCR diagnosis. The performance of the MAP assay is comparable to that of the IHP assay and to those in previously reported studies evaluating commercial tests (galactomannan enzyme-linked immunosorbent assay).