In our research, a series of toxicity tests with the underlying toxic mechanisms were conducted with Trifolium pratense L. (red clover). After a different silver exposing time (e.g., 1 d, 3 d, and 8 d), noticeable alterations had occurred in root elongation, photosynthetic pigments, ion leakage, cell death, reactive oxygen species (ROS), superoxide dismutase (SOD), peroxidase (POD), catalase (CAT), ascorbate peroxidase (APX), glutathione peroxidase (GPX), glutathione reductase (GR), glutathione (GSH), and ascorbate (AsA) assays. On average, the root tissues of red clover retained on approximately 84.59 % of the total Ag absorbed as a tolerance strategy to Ag⁺ stress. Ultrastructural damages in morphological characteristics were observed and speculated to be related to severe free radical attack induced by Ag⁺ and argentiferous NPs synthesis in vivo. Whole genome resequencing detected 1, 900, 266 and 1, 996, 263 single nucleotide variants (SNVs) and 868, 643 and 924, 957 insertions and deletions (InDels) for the two experimental plants (i.e., 4-day treated and 8-day treated) compared to controls. In addition, genomic resequencing data revealed structural variations (SVs) and copy number variations (CNVs) in experimental groups under Ag⁺ stress. Gene ontology (GO) enrichment analysis for genes containing SNVs indicated that their molecular functions were mostly related to DNA binding transcription factor activity. These findings might provide a critical insight into the regulatory pathways of Ag⁺ affected plants.