Potato is globally recognized as the fourth most crucial staple food crop, trailing behind wheat, rice, and maize. Cadmium (Cd), a predominant heavy-metal pollutant in agricultural soils, demonstrates high biological toxicity and mobility. Therefore, exploring the genetic and molecular mechanisms underpinning cadmium tolerance in potato is of substantial theoretical and practical significance. In this research, an F₂ population composed of 170 families was established through the cross-breeding of homozygous diploid potato lines HD-5 (highly cadmium-tolerant) and M9 (cadmium-sensitive). Employing hydroponic cultivation, six traits, namely plant height (PH), root length (RL), shoot fresh weight (SFW), root fresh weight (RFW), chlorophyll content (SPAD), and nitrogen content (LNC), were measured in potato seedlings following a 9-day treatment with 40 mg·L⁻¹ CdCl₂. By utilizing the high-density genetic map of this population for QTL mapping, a total of 35 genetic loci associated with cadmium tolerance in potato seedlings were identified. Notably, loci21 and loci22 on chromosome 9, loci29 on chromosome 10, and loci31 and loci33 on chromosome 12 were consistently detected across multiple environmental conditions. This reproducibility across environments suggests the phenotypic stability of these five loci, which are thus considered reliable and robust genetic determinants. In addition, transcriptome sequencing analysis of roots from parental lines HD-5 and M9 after cadmium treatment revealed that significantly differentially expressed genes between the two parents were associated with glutathione metabolism and photosynthesis. By integrating QTL mapping, transcriptome analysis, and gene annotation, we screened four candidate genes involved in cadmium tolerance regulation: DM8C09G01000 (GST), DM8C09G01060 (GST), DM8C09G02130 (OXP1), and DM8C06G22960 (PsaH). These findings provide molecular targets and a genetic basis for molecular breeding of cadmium-tolerant potato varieties.