Salt stress significantly constrains plant growth and development, with the root system serving as the primary organ for sensing and responding to salinity. Poplar (Populus spp.), a widely cultivated tree species, plays important roles in urban greening, windbreaks and sand fixation, and various economic sectors. In this study, we conducted a comparative analysis of the salt tolerance of root systems of six different poplar cultivars. Key physiological indicators—including root activity, ion homeostasis, reactive oxygen species (ROS)-scavenging capacity, and nitric oxide (NO) content—were evaluated under saline conditions. Our findings indicate that the salt resistance of the six cultivars follows the order: P. simonii > P. cathayana > P. tremula × P. alba ‘717’ > P. deltoides × P. euramericana ‘Nanlin 895’ > P. deltoides × P. euramericana ‘Bofeng 3’ > P. alba × P. glandulosa ‘84K’. Furthermore, a transcriptomic analysis was performed on the most resilient species, P. simonii. These results reveal that P. simonii primarily orchestrates its salt stress response through the modulation of MAPK cascades and nitrogen metabolism pathways. This research provides theoretical support for elucidating the molecular mechanisms underlying the salt stress response in poplars and offers a foundation for the molecular breeding of salt tolerant poplar varieties.