Tobacco cultivars greatly differ in morphology, size and regional growing conditions. Usually, they are identified on the basis of the phenotypical characteristics and marketing grade of the leaf. Molecular methods have so far played a lesser role but a combination of molecular analyses can allow to answer any question on genetic identification between two or more organisms. In tobacco, as in any higher eukaryote, ribosomal RNA genes (rDNA) are highly reiterated and organised in tandem arrays at the nucleolar organising regions of chromosomes. Each repeating unit usually contains one coding sequence for each of the 18S, 5.8S and 28S rRNAs, spaced out three intergenic sequences from one another. The transcribed sequences, as well as the intergenic DNA (IGS) between each transcription unit, are highly variable in length and primary structure among organisms and even among individuals. Being such regions virtually free of any functional constraints, Internal Transcribed Spacers (ITS) and IGS sequences can evolve at a faster rate, as reflected in the number of substitutions, point mutations and well documented length variations; these characteristics allow to perform identification and population studies. Purpose of this study is to describe the characteristics of the Internal Transcribed Spacers of cultivars of Nicotiana tabacum , in order to attempt the identification of any tobacco cultivars. Nucleotide sequences of the nuclear ribosomal DNA ITS and short stretches of the adjacent 5.8S coding region were determined in different tobacco cultivars. ITS regions were directly sequenced from Polymerase Chain Reaction (PCR) amplified DNA fragments. The possible use of the level of variation within ITS regions for the identification of single, or groups of, tobacco cultivars on local or regional scale is discussed.