F-box proteins are generally responsible for substrate recognition in the Skp1-Cullin-F-box complexes that are involved in protein degradation via the ubiquitin-26S proteasome pathway. In plants, F-box genes influence a variety of biological processes, such as leaf senescence, branching, self-incompatibility, and responses to biotic and abiotic stresses. The number of F-box genes in Populus ( Populus trichocarpa; approximately 320) is less than half that found in Arabidopsis ( Arabidopsis thaliana; approximately 660) or Oryza ( Oryza sativa; approximately 680), even though the total number of genes in Populus is equivalent to that in Oryza and 1.5 times that in Arabidopsis. We performed comparative genomics analysis between the woody perennial plant Populus and the herbaceous annual plants Arabidopsis and Oryza in order to explicate the functional implications of this large gene family. Our analyses reveal interspecific differences in genomic distribution, orthologous relationship, intron evolution, protein domain structure, and gene expression. The set of F-box genes shared by these species appear to be involved in core biological processes essential for plant growth and development; lineage-specific differences primarily occurred because of an expansion of the F-box genes via tandem duplications in Arabidopsis and Oryza. The number of F-box genes in the newly sequenced woody species Vitis ( Vitis vinifera; 156) and Carica ( Carica papaya; 139) is similar to that in Populus, supporting the hypothesis that the F-box gene family is expanded in herbaceous annual plants relative to woody perennial plants. This study provides insights into the relationship between the structure and composition of the F-box gene family in herbaceous and woody species and their associated developmental and physiological features.