Members of the plant-specific WOX (WUSCHEL-related homeobox) transcription factor family have been reported to play important roles in peptide signaling that regulates stem cell maintenance and cell fate specification in various developmental processes. Even though remarkable advances have been made in studying WOX genes in Arabidopsis, little is known about this family in Solanaceae species. A total of 45 WOX members from five Solanaceae species were identified, including eight members from Solanum tuberosum, eight from Nicotiana tomentosiformis, 10 from Solanum lycopersicum, 10 from Nicotiana sylvestris and nine from Nicotiana tabacum. The newly identified WOX members were classified into three clades and nine subgroups based on phylogenetic analysis using three different methods. The patterns of exon-intron structure and motif organization of the WOX proteins agreed with the phylogenetic results. Gene duplication events and ongoing evolution were revealed by additional branches on the phylogenetic tree and the presence of a partial WUS-box in some non-WUS clade members. Gene expression with or without CLE (clavata3 (clv3)/embryo surrounding region-related) peptide treatments revealed that tobacco WOX genes showed similar or distinct expression patterns compared with their Arabidopsis homologues, suggesting either functional conservation or divergence. Expression of Nicotiana tabacum WUSCHEL (NtabWUS) in the organizing center could rescue the wus-1 mutant phenotypes in Arabidopsis, implying conserved roles of the Solanaceae WOX proteins in peptide-mediated regulation of plant development.