Calmodulin-binding transcription activators (CAMTAs) represent the novel gene family of transcriptional regulators, which play important biological functions. Though, the first ever plant CAMTA gene was evidenced in Nicotiana tabacum in 2002. But, the systematic identification, origin and function of this gene family has not been performed due to the lack of reference genome information until now. Here, we identified 29 CAMTA genes in four Nicotiana species, including thirteen NtabCAMTAs, six NsylCAMTAs, and five NtomCAMTAs and NbenCAMTAs. These CAMTA families were classified into five phylogenetic groups (I-V), among which, the group-IV CAMTAs probably emerged the earliest. The NtabCAMTA family genes have diverse structures, and are randomly localized on five chromosomes and scaffolds. N. tabacum acquired 11 copies of homolog CAMATA genes from the parental genomes of N. tomentosiformis and N. sylvestris, followed by expansion through polyploidization and duplication. The NtabCAMTA genes were differentially expressed in different plant parts, and showed sensitivity towards different abiotic and biotic stresses. Co-expression network analysis revealed that some NtabCAMTA subunits interact with each other, and co-expressed. The current study is the first report presenting a comprehensive overview of Nicotiana CAMTA families, and opens a new avenue for the improvement of the cultivated tobacco.