In the past few years, many studies have reported that the transcription factor Nuclear Factor Y (NF-Y) gene family plays important roles in embryonic development, photosynthesis, flowering time regulation and stress response, in various plants. Although the NF-Y gene family has been systematically studied in many species, little is known aboutNF-Ygenes in Populus. In this study, the NF-Y gene family in the Populus genome was identified and its structural characteristics were described. Fifty-twoNF-Ygenes were authenticated in thePopulus trichocarpagenome and categorized into three subfamilies (NF-YA/B/C) by phylogenetic analysis. Chromosomal localization of these genes revealed that they were distributed randomly across 17 of the 19 chromosomes. Segmental duplication played a vital role in the amplification of Populus NF-Y gene family. Moreover, microsynteny analysis indicated that, amongPopulus trichocarpa,Arabidopsis thaliana,Vitis viniferaandCarica papaya,NF-Yduplicated regions were more conserved betweenPopulus trichocarpaandVitis vinifera. Redundant stress-relatedcis-elements were also found in the promoters of most 13NF-YAgenes and their expression levels varied widely following drought, salt, ABA and cold treatments. Subcellular localization experiments in tobacco showed thatPtNF-YA3was localized in nucleus and cytomembrane, while PtNF-YA4 was only in the nucleus in tobacco. According to the transcriptional activity experiments, neither of them had transcriptional activity in yeast. In summary, a comprehensive analysis of the Populus NF-Y gene family was performed to establish a theoretical basis for further functional studies on this family.