Mitogen-activated protein kinase kinase kinases (MAPKKK, MAP3K, MKKK or MEKK) regulate plant growth and development and are involved in regulatory pathways for plant resistance to a wide range of biotic and abiotic stresses. However, the phylogeny of the MAPKKK gene family and the biological function of VaMAPKKK15 under low-temperature stress in Vitis vinifera (V. vinifera) and Vitis amurensis (V. amurensis) are not known. In this study, members of the V. amurensis and V. vinifera MAPKKK gene families were identified and analyzed, and a total of 67 VvMAPKKKs and 59 VaMAPKKKs were identified. Phylogenetic analysis showed that this family of genes was divided into three subfamilies, Raf, MEKK and ZIK. In addition, VaMAPKKK15 self-activation validation showed that VaMAPKKK15