SUP gene family expression and regulation patterns reported in dioecious woody plant. Phylogenetic and nucleotide diversity analysis indicated PtoSUP1 is highly conserved and has undergone strong purifying selection. The molecular basis of SUPERMAN (SUP) regulation during floral development in monoecious plants has been extensively studied, but little is known of the SUP gene family in dioecious woody plants. In this study, we systematically examined the diversification of the SUP gene family in Populus, integrating genomic organization, expression, and phylogeny data. SUP family members showed sex-specific expression throughout flower development. Transcript profiling of rare gynomonoecious poplar flowers revealed that a significant reduction in PtoSUP1 mRNA might be important for stamen development in gynomonoecious poplar flowers. We found that the coding regions of Populus SUP genes are very highly conserved and that synonymous sites in exon regions have undergone strong purifying selection during SUP evolution in Populus. These results indicate that SUP genes play an important role in floral development of dioecious plants. Expression analysis of SUP suggested possible regulatory mechanisms for gynomonoecious poplar flower development. These findings provide an important insight into the mechanisms of the evolution of SUP function and may help enable engineered regulation of flower development for breeding improved tree varieties.