Kentucky bluegrass (Poa pratensis L.) is a widely utilized cool-season turfgrass species in landscape architecture and ecological restoration projects. Nevertheless, the majority of commercially available cultivars currently exhibit insufficient drought tolerance, necessitating frequent irrigation during hot and arid seasons to sustain optimal turf performance and aesthetic quality. Here, we established a reliable chromosome doubling protocol for octaploid (8X) Kentucky bluegrass seeds, successfully generating hexadecaploid (16X) progeny. Drought stress experiments were conducted to evaluate the stress resistance of the polyploid lines, and the results demonstrated that 16X plants displayed significantly enhanced drought tolerance relative to their 8X counterparts. After drought stress, the 16X plants exhibited significantly higher relative water content, chlorophyll content, superoxide dismutase (SOD) activity, catalase (CAT) activity, soluble sugar content, and free proline content than the 8X plants, whereas malondialdehyde (MDA) content and electrolyte leakage (EL) were significantly lower than those of the 8X plants.To elucidate the molecular mechanisms underlying the improved drought tolerance in 16X Kentucky bluegrass, transcriptome sequencing was performed. Bioinformatic analysis revealed that differentially expressed genes (DEGs) were significantly enriched in three key metabolic and signaling pathways, namely plant-pathogen interaction, carbon metabolism, and plant hormone signal transduction. In addition, we performed in vitro protein pull-down assays to explore the pathways regulated by a 143-3 protein of interest that may be associated with drought stress. To the best of our knowledge, this study represents the first systematic report on the artificial induction of polyploidy in Kentucky bluegrass via chromosome doubling. This innovative breeding strategy not only provides a promising approach to broaden the application of Kentucky bluegrass in arid and semi-arid regions but also holds significant potential for reducing irrigation-related costs in municipal landscape management practices.