Protein kinases play an important role in every aspect of cellular life.  In this study, we systemically identified protein kinases from the predicted proteomes of 59 representative fungi from Ascomycota and Basidiomycota.  Comparative analysis revealed that fungi from Ascomycota and Basidiomycota differed in the number and variety of protein kinases.  Some groups of protein kinases, such as calmodulin/calcium regulated kinases (CMGC) and those with the highest group percentages are the most prevalent protein kinases among all fungal species tested.  In contrast, the STE group (homologs of the yeast STE7, STE11 and STE20 genes), was more abundant in Basidiomycetes than in Ascomycetes.  Importantly, the distribution of some protein kinase families appeared to be subphylum-specific.  The tyrosine kinase-like (TKL) group had a higher protein kinase density in Agaricomycotina fungi.  In addition, the distribution of accessory domains, which could have functional implications, demonstrated that usage bias varied between the two phyla.  Principal component analysis revealed a divergence between the main functional domains and associated domains in fungi.  This study provides novel insights into the variety and expansion of fungal protein kinases between Ascomycota and Basidiomycota.