Abstract: Expressed sequence tags (EST) are widely used in gene function research and molecular marker development. In order to obtain a large number of EST sequences of tobacco, a variety of tissues and organs from Nicotiana tomentosiformis and Nicotiana sylvestris were taken as plant materials, and two full-length enriched cDNA libraries were constructed using the CloneMiner cDNA method. The EST sequences were used for sequence assembly, functional annotation, phylogenetic analysis and molecular marker development. The normalized full-length cDNA libraries were constructed successfully from Nicotiana tomentosiformis and Nicotiana sylvestris. The titer were 0.72×10⁶ and 1.12×10⁶ pfu/mL, respectively, and the recombination rates were approximately 94% and 93%, respectively. The average length of inserted cDNA fragments was 1.4 kb. 20 953 ESTs were generated from the full-length enriched cDNA libraries, and assembled into 10 504 unigenes. All of the ESTs from allopolyploid tobacco (Nicotiana tabacum) and its two diploid progenitors, Nicotiana tomentosiformis and Nicotiana sylvestris were assembled, resulting in 34 450 contigs and 123 511 singletons. The global assembly showed that the transcripts from the resident T- and S-genomes in the allotetraploid nucleus were more closely related to their diploid homologs than they were to each other. In total, 104 915 coding sequences were identified, of which 73 670 sequences contained functional domains. Approximately 81% of the unigenes had homologs in tomato. Furthermore, we found 11 869 putative simple sequence repeats (SSR) and 55 369 single nucleotide polymorphisms (SNPs). The EST resources have important implications for gene function research and molecular breeding.