Two pairs of degenerate primers were designed according to the conserved amino acid sequences of several mitogen-activated protein kinase kinase kinases (MAPKKK) from basidiomycetes. Based on the conserved DNA sequence, which was obtained by nested degenerate PCR, a whole DNA sequence of MAPKKK of Volvariella volvacea (VV-MAPKKK) was obtained by blasting with Volvariella volvacea genome. VV-MAPKKK gene, containing 4 introns and codeing for a 1405 aa protein (VV-MAPKKK), is 4434 bp in length. The similarity of VV-MAPKKK compared with Hog-MAPKKK protein from Cryptococcus neoformans, Paracoccidioides brasiliensis and Cochliobolus heterostrophus is 58%, 57% and 56%, respectively. Phylogenetic analysis showed that VV-MAPKKK and Hog-MAPKKK protein homolog belong to the same clade. These data supported that VV-MAPKKK protein is a Hog-MAPKKK protein homolog from Volvariella volvacea.