HwHog1A/B, Hortaea werneckii homologues of the MAP kinase Hog1 from Saccharomyces cerevisiae, are vital for the extreme halotolerance of H. werneckii. In mesophilic S. cerevisiae, Hog1 is phosphorylated already at low osmolyte concentrations, and regulates expression of a similar set of genes independent of osmolyte type. To understand how HwHog1 kinases activity is regulated in H. werneckii, we studied HwHog1A/B activation in vivo, by following phosphorylation of HwHog1A/B in H. werneckii exposed to various osmolytes, and in vitro, by measuring kinase activities of recombinant HwHog1A, HwHog1B and Hog1ΔC. To this end, highly pure and soluble recombinant Hog1 homologues were isolated from insect cells. Our results demonstrate that HwHog1A/B are, in general, transiently phosphorylated in cells shocked with ≥3 M osmolyte, yet constitutive phosphorylation is observed at extreme NaCl and KCl concentrations. Importantly, phosphorylation profiles differ depending on the osmolyte type. Additionally, phosphorylated recombinant HwHog1A/B show lower specific kinase activities compared to Hog1ΔC. In summary, HOG pathway MAPKs in the extremely halotolerant H. werneckii show unique characteristics compared to S. cerevisiae homologues. The reported findings contribute to defining the key determinants of H. werneckii osmotolerance, which is important for its potential transfer to economically relevant microorganisms and crops.