Cell-wall invertase genes are spatially and temporally regulated in several plant species, including Daucus carota L., Lycopersicon esculentum L. and Solanum tuberosum L. However, few studies of cell-wall invertase genes of trees have been conducted, despite the importance of trees as a source of lignocellulosic biopolymers.We identified three putative cell-wall invertase genes in hybrid poplar (Populus alba L. × grandidentata Michx.) that showed higher homology to each other than to cell-wall invertases of other dicotyledonous species, with two of the genes (Pa×gINV2 and Pa×gINV3) appearing as a genomic tandem repeat. These genes are more similar to each other than to tandemly repeated cell-wall invertases of other plants, perhaps indicating parallel evolution of a duplication event with cell-wall invertases in dicotyledons. Spatial and temporal expression analyses throughout a complete annual cycle indicated that Pa×gINV1 and Pa×gINV2 are highly regulated in vegetative tissues during three distinct growth phases: early growth, dormancy and post-dormancy. Expression of the third gene (Pa×gINV3) appears to be tightly regulated and may represent a floral-specific cell-wall invertase. Of the two genes expressed in vegetative tissues, Pa×gINV1 appears to be exclusively involved in processes related to dormancy, whereas Pa×gINV2 appears to encode an enzyme involved in phloem unloading and in providing actively growing tissues, such as developing xylem, with the energy and carbon skeletons necessary for respiration and cell wall biosynthesis.