Based on multiple sequence alignment of mitogen activated protein kinase (MAPK) genes from 28 fungal species, we could identify twelve new hallmark sequences, specific to YSAPK (yeast and fungal stress activated protein kinases) MAPK subgroup within fungal MAPKs. Six of the motifs (I-a, I-b, IV-b, V-b, X-b1 and X-b2) showed especially high degree of specificity. Two of these six motifs, I-b (SA[RK]DQLT) and IV-b (F[IL]SPLED[IV]) were specific within eukaryotic proteins, too. The other type of motifs contained not only YSAPK specific residues but residue(s) conserved on fungal MAPK and all MAPK levels. From the viewpoint of functional role, YSAPK motif VII-a (IL[VI]NENCDL) coincided with a loop spanning 7b-8b sheets in human p38a and ERK2 MAPK proteins (consensus indicated by bold face letters). This motif was shown to be involved in interaction with L-x-L type docking motifs of activators (MAPK kinases) and transcription factors. A fungal MAPK specific signal sequence in protein kinase subdomain IX-b (AE[ML][LVI]xG[KR]PxFxG[KR]D) was also described. A subgroup specific nested PCR-based cloning approach was developed to amplify YSAPK sequences in different filamentous fungal species based on motifs I, I-b, VIII and X-b3 as primers. Putative YSAPK MAPK amplicons obtained by this approach were identified by (i) the presence of newly characterized YSAPK specific motifs and (ii) alignments to known YSAPK MAPK genes. A neighbor-joining phylogenetic tree constructed by comparing 50 fungal MAPK sequences clearly demonstrated a dichotomic origin of fungal MAPKs and separated YSAPK MAPKs from the other two fungal MAPK subgroups. Comparing the number and distribution of known MAPK genes in Fusarium and other species it is tempting to speculate that this triple structural diversity of MAPKs, at least in filamentous species is a general phenomenon. Phylogenetic analysis revealed no separation of phytopathogenic species. Blumeria graminis, Alternaria brassicicola and Aspergillus species, however were separated from the majority of filamentous Ascomycetes in all of the three MAPK subgroups.