Potato (Solanum tuberosum L.) is the third most important food crop worldwide after wheat and rice in terms of human consumption.  A critical domestication trait for potato was the decrease of toxic steroidal glycoalkaloids (SGAs) in tuber flesh.  Here, we used a diploid F2 segregating population derived from a cross between S. tuberosum and the wild potato species Solanum chacoense to map the quantitative trait loci (QTLs) associated with the regulation of SGAs content in tuber flesh.  In a three-year study, we identified two QTLs on chromosomes 2 and 8 affecting SGAs content in tuber flesh.  The QTL on chromosome 8 harbors 38 genes that are co-expressed with the GLYCOALKALOID METABOLISM genes.  These findings lay the foundation for exploiting the genes controlling SGAs content in tuber flesh and they provide a theoretical basis for the use of wild germplasm in potato breeding.