A WUSCHEL-related homeobox (WOX) gene family has been implicated in promoting vegetative organs to embryonic transition and maintaining plant embryonic stem cell identity. Using genome-wide analysis, we identified 17 candidates, WOX genes in ramie (Boehmeria nivea). The genes (BnWOX) showed highly conserved homeodomain regions typical of WOX. Based on phylogenetic analysis, they were classified into three distinct groups: modern, intermediate, and ancient clades. The genes displayed 65% and 35% collinearities with their Arabidopsis thaliana and Oryza sativa ortholog, respectively, and exhibited similar motifs, suggesting similar functions. Furthermore, four segmental duplications (BnWOX10/14, BnWOX13A/13B, BnWOX9A/9B, and BnWOX6A/Maker00021031) and a tandem-duplicated pair (BnWOX5/7) among the putative ramie WOX genes were obtained, suggesting that whole-genome duplication (WGD) played a role in WOX gene expansion. Expression profiling analysis of the genes in the bud, leaf, stem, and root of the stem cuttings revealed higher expression levels of BnWOX10 and BnWOX14 in the stem and root and lower in the leaf consistent with the qRT-PCR analysis, suggesting their direct roles in ramie root formation. Analysis of the rooting characteristics and expression in the stem cuttings of sixty-seven different ramie genetic resources showed a possible involvement of BnWOX14 in the adventitious rooting of ramie. Thus, this study provides valuable information on ramie WOX genes and lays the foundation for further research.