Delayed diagnosis in invasive aspergillosis (IA) contributes to its high mortality. Gliotoxin (GT) and bis-methyl-gliotoxin (bmGT) are secondary metabolites produced by Aspergillus during invasive, hyphal growth and may prove diagnostically useful. Because IA pathophysiology and GT's role in virulence vary depending on the underlying host immune status, we hypothesized that GT and bmGT production in vivo may differ in three mouse models of IA that mimic human disease. We defined temporal kinetics of GT and bmGT in serum, bronchoalveolar lavage fluid (BALF) and lungs of A. fumigatus-infected chronic granulomatous disease (CGD), hydrocortisone-treated, and neutropenic mice. We harvested lungs for assessment of fungal burden, histology and GT/bmGT biosynthetic genes' mRNA induction. GT levels were higher in neutropenic versus CGD or steroid-treated lungs. bmGT was persistently detected only in CGD lungs. GT, but not bmGT, was detected in 71% of sera and 50% of BALF of neutropenic mice; neither was detected in serum/BALF of CGD or steroid-treated mice. Enrichment of GT in Aspergillus-infected neutropenic lung correlated with fungal burden and hyphal length but not induction of GT biosynthetic genes. In summary, GT is detectable in mouse lungs, serum and BALF during neutropenic IA, suggesting that GT may be useful to diagnose IA in neutropenic patients.