The traditional approach to potato breeding, the classical “mate and phenotype” approach is relatively costly and because phenotyping and growth capacity is limited, this are being slowly replaced by Marker Assisted Selection (MAS) breeding schemes. MAS is based on the presence of DNA polymorphic markers closely associated with trait of interest and as such allows screening of much larger effective populations in a cost-effective manner already at the seed or seedling stages. However, it requires discovery and development of relevant molecular markers, which up to recently has been both costly and time consuming process. Here, a novel method for Quantitative Trait Locus (QTL) analysis has been developed, that allows for development of specific markers by use of genomic sequence reads and the recently published reference genome sequence for potato. Prior to sequencing the mapping population is phenotyped for the trait of interest and contrasting bulks of individuals are formed. Following validation on a simulated dataset, the new method was applied to a mapping population of 186 dihaploid potato clones originating from a cross between 90-HAF-01 (S. tuberosum1) and 90-HAG-15 (S.tuberosum2 x S.sparsipilum), phenotyped for water use efficiency. This population has also previously been phenotyped for the total glycoalkaloid (TGA) content.