Soil salinity is an important limiting factor to tree growth and productivity. Populus alba L. is a moderately salt-tolerant species and its natural populations are adapted to contrasting environments, thus providing genetic resources to identify key genes for tolerance to abiotic stress, such as salinity. To elucidate the molecular and genetic basis of variation for salinity tolerance in P. alba, we analyzed the short-term ecophysiological and transcriptome response to salinity. Two contrasting genotypes, 6K3, salt sensitive, and 14P11, salt tolerant, originating from North and South Italy, respectively, were challenged with salt stress (200 mM NaCl). Sodium accumulated in the leaves of salt-treated plants and its concentration increased with time. The net photosynthesis was strongly reduced by salinity in both genotypes, with 6K3 being significantly more affected than 14P11. The transcriptional changes in leaves were analyzed using cDNA microarrays containing about 7000 stress-related poplar expressed sequence tags (EST). A microarray experiment based on RNA pooling showed a number of salinity-regulated transcripts that markedly increased from 3 h to 3 days of salinity treatment. Thus, a detailed analysis was performed on replicated plants collected at 3 days, when ∼20% of transcripts showed significant change induced by salinity. In 6K3, there were more genes with decreased expression than genes with increased expression, whereas such a difference was not found in 14P11. Most transcripts with decreased expression were shared between the two genotypes, whereas transcripts with increased expression were mostly regulated in a genotype-specific manner. The commonly decreased transcripts (71 genes) were functionally related to carbohydrate metabolism, energy metabolism and photosynthesis. These biological processes were consistent with the strong inhibition of photosynthesis, caused by salinity. The commonly increased transcripts (13 genes) were functionally related to primary metabolism and biosynthesis of proteins and macromolecules. The salinity-increased transcripts discriminated the molecular response of the two genotypes. In 14P11, the 21 genes specifically salinity-induced were related to stress response, cell development, cell death and catabolism. In 6K3, the 15 genes with salinity-increased expression were involved in protein biosynthesis, metabolism of macromolecules and cell organization and biogenesis. The difference in transcriptome response between the two genotypes could address the molecular basis of intra-specific variation of salinity tolerance in P. alba.