n vitro evaluation of salinity effects on male aspen clone W52 (Populus tremula L.) and the hybrid aspen clone T89 (Populus tremula L. × Populus tremuloides ‘Michx’) were investigated after seawater treatments (control, and ‘concentrations’ of 8.0, 12.0 and 14.0 dS/m). A salinity level of 14.0 dS/m was found to be lethal. All the vegetative parameters of both Populus clones were significantly decreased at 12.0 dS/m. The amounts of pigments and percentages of K+ and Na+, as well as peroxidase activity in the plantlets, were determined as indicators for salt stress tolerance. Also, a genetic diversity study between the two Populus clones using inter-simple sequence repeat (ISSR)-PCR analysis showed specific marker bands for the W52 clone with high molecular sizes: 1900, 1500, 1150, and 1000 bp. Furthermore, the level of transcription factor homeodomain-leucine zipper III_1 (PagHD-ZIPIII_1) gene transcripts was positively affected in response to salt stress and influenced by genotypes. Hence, the response of the two clones to salt stress was associated with the up-regulation of PagHD-ZIPIII_1 gene which could be a good candidate for in vitro selection for salt tolerance genotypes. Generally, the clone T89 is more salinity tolerant compared to the clone W52.