The genome of the osmophilic Aspergillus wentii, unlike that of the osmotolerant Aspergillus nidulans, contains only the gfdA but not the gfdB glycerol 3-phosphate dehydrogenase gene. Here, we studied transcriptomic changes of A. nidulans (reference strain and DgfdB gene deletion mutant) and A. wentii (reference strain and An-gfdB expressing mutant) elicited by high osmolarity. A. nidulans showed canonic hyperosmotic stress response characterized by upregulation of trehalose and glycerol metabolism genes (including gfdB) as well as genes of the high-osmolarity glycerol (HOG) map kinase pathway. Deletion of gfdB caused only negligible alterations in the transcriptome suggesting that the glycerol metabolism was flexible enough to compensate for the missing GfdB activity in this species. A. wentii responded differently to increased osmolarity than A. nidulans: E.g. bulk upregulation of glycerol and trehalose metabolism genes as well as HOG pathway genes were not detected. Expression of An-gfdB in A. wentii did not abolish osmophilia, but it reduced growth and caused much bigger alterations in the transcriptome than the missing gfdB gene did in A. nidulans. Flexible glycerol metabolism and hence two differently regulated gfd genes may be more beneficial for osmotolerant (living under changing osmolarity) than for osmophilic (living under constantly high osmolarity) species. Overall design: Comparative gene expression profiling analysis of RNA-seq data for mycelia of A. nidulans (reference strain and DgfdB gene deletion mutant) and Aspergillus wentii (reference strain and A. nidulans-gfdB expressing mutant) at normal osmolarity, in the presence of 1 M sorbitol, 2 M NaCl, or 1 M sorbitol + 2 M NaCl with three biological replicates.