Background: Brachypodium distachyon is emerging as a widely recognized model plant that has very close relations with several economically important Poaceae species.MAPK cascade is known to be an evolutionarily conserved signaling module involved in multiple stresses.Although the gene sequences of MAPK and MAPKK family have been fully identified in B.distachyon,the information related to the upstream MAPKKK gene family and,especially the regulatory network among MAPKs,MAPKKs and MAPKKKs upon multiple stresses remains to be understood.Results: In this study,we have identified MAPKKKs which belong to the biggest gene family of MAPK cascade kinases.We have systematically investigated the evolution of whole MAPK cascade kinase gene family in terms of gene structures,protein structural organization,chromosomal localization,orthologs construction and gene duplication analysis.Our results showed that most BdMAPK cascade kinases were located at the low-CpG-density region,and the clustered members in each group shared similar structures of the genes and proteins.Synteny analysis showed that 62 or 21 pairs of duplicated orthologs were present between B.distachyon and Oryza sativa,or between B.distachyon and Arabidopsis thaliana respectively.Gene expression data revealed that BdMAPK cascade kinases were rapidly regulated by stresses and phytohormones.Importantly,we have constructed a regulation network based on co-expression patterns of the expression profiles upon multiple stresses performed in this study.Conclusions: BdMAPK cascade kinases were involved in the signaling pathways of multiple stresses in B.distachyon.The network of co-expression regulation showed the most of duplicated BdMAPK cascade kinase gene orthologs and their convergent function,whereas few of them developed divergent function in the evolutionary process.The identified MAP3K family genes and the constructed MAPK cascade regulation network under multiple stresses provide valuable information for further investigation of the functions of BdMAPK cascade kinase genes.