Signal transduction pathways play crucial roles in cellular adaptation to environmental changes. In this study, we employed comparative genomics to analyse the high osmolarity glycerol pathway in fungi. This system contains several signalling modules that are used throughout eukaryotic evolution, such as a mitogen-activated protein kinase and a phosphorelay module. Here we describe the identification of pathway components in 20 fungal species. Although certain proteins proved difficult to identify due to low sequence conservation, a main limitation was incomplete, low coverage genomic sequences and fragmentary genome annotation. Still, the pathway was readily reconstructed in each species, and its architecture could be compared. The most striking difference concerned the Sho1 branch, which frequently does not appear to activate the Hog1 MAPK module, although its components are conserved in all but one species. In addition, two species lacked apparent orthologues for the Sln1 osmosensing histidine kinase. All information gathered has been compiled in an MS Excel sheet, which also contains interactive visualisation tools. In addition to primary sequence analysis, we employed analysis of protein size conservation. Protein size appears to be conserved largely independently from primary sequence and thus provides an additional tool for functional analysis and orthologue identification.