microRNAs (miRNAs) are a recently discovered class of small (~21 nt) endogenous gene regulators that have been shown to play an important role in plant growth and development by aiding in organ maturation, hormone signaling, tissue differentiation, and plant tolerance to environmental stress. Since a list of miRNAs has never been generated for tobacco, we employed genome survey sequence analysis to computationally identify 259 potentially conserved tobacco miRNAs, belonging to 65 families, and validated 11 of these miRNAs using qRT-PCR. The 65 miRNA families were dramatically different in size. miRNA precursor (pre-miRNA) sequence analysis showed that tobacco pre-miRNAs greatly varied from 45 to 635 nt in length with an average of 141 ± 108 nt. We were also able to determine the presence of antisense miRNAs as well as miRNA clusters in tobacco. Using previously established protocols, a total of 1,225 potential target genes were predicted for the newly identified tobacco miRNAs. These target genes include transcription factors, DNA replication proteins, metabolic enzymes, as well as other gene targets necessary for proper plant maturation. The results of this study show that conserved miRNAs exist in tobacco and suggest that these miRNAs may play an important role in tobacco growth and development.