The CLE family (CLAVATA3/embryo surrounding region-related), a class of small secreted proteins, play important roles in plant development and stress responses. Members of the CLE family have been characterized in a number of plant species, including Arabidopsis and rice. However, limited information is available about CLE peptides in tobacco (Nicotiana tabacum) and related Nicotiana species. Here we report the identification of 84 CLE family members in three Nicotiana species based on sequence similarity. The newly identified CLE members, including 41 from N. tabacum, 19 from N. sylvestris, and 24 from N. tomentosiformis, together with 32 CLEs from Arabidopsis and 52 CLEs from tomato, formed 9 subgroups in a phylogenic tree. The unbalanced distribution of the Nicotiana CLEs in the subgroups suggested potential preferential gene family expansion during evolution. Expression of the NtCLE genes was analyzed and a number of the NtCLEs showed induced expression upon abiotic stress treatments. Synthetic peptides of several NtCLEs, when applied to detached tobacco leaf discs, were able to increase plants’ tolerance to osmotic and salinity stresses, suggesting potential roles of CLE peptides in the stress responses of tobacco.