Potato cultivars (Solanum tuberosum L.) are delicate to cold and frost. Low temperatures have a negative impact on potato growth, development and yield. However, genetic detection of frost tolerance loci is currently lacking. Here, we report a BC1 population that was constructed by crossing the diploid cold-resistant wild species S. commersonii with the diploid cold-sensitive wild species S. verrucosum. The frost tolerance of the resulting offspring was determined through four years of natural field frost experiments. Together with bulked segregant analysis (BSA) technology and traditional quantitative trait locus (QTL) mapping, four highly related QTLs were detected on Chr02 and Chr11. Furthermore, we constructed a partial genetic linkage map of Chr02, and two QTLs (QTL1 and QTL2) were detected on Chr02. Four genes located in candidate intervals have been reported to be associated with tolerance to abiotic stress and were significantly differentially expressed among the resistant and sensitive. Taken together, our results also suggest that the frost resistance of plants under variable low-temperature conditions is controlled by different loci.