The efficiency of the Bulk Segregant Analysis (BSA) had clearly been demonstrated to detect genomic regions and genes involved in diverse traits. It allows large experiments reducing the cost and time and preserving the power of full individual's population analysis. These past few years the combination of BSA and New Generation Sequence (NGS) data (BSA-Seq) gave a new accuracy and depth to the discovery on many traits of interest, mainly on crop and model species. In our study, we applied the BSA-Seq in a heterozygous and diploid genome context. We worked on the progenies derived from an interspecific cross Populus deltoides x Populus trichocarpa in which segregates the resistance to Melampsora larici populina (Mlp) leaf rust. We detected DNA variations with freebayes/0.9.21 and the soft masked genome of Populus trichocarpa Nisqually v3.0 as reference. Comparing DNA variations in between parents and bulks we obtained 27 regions or Quantitative Trait Loci based on NGS analysis (QTL-Seq) which could explain the resistance to Mlp. We first evaluated the strategy retrieving a previously cloned Mlp resistance gene governing the uredinia size in Populus trichocarpa clone 101-74 (RUS). Then we identified genomic markers which should better characterize this locus. So we demonstrated that, in our context, BSA-Seq allows us to improve the fine mapping of a major gene. We think it can be a promising method on a high heterozygous diploid genome as Poplar, to decipher complex trait. Next step is to proceed with it to fine map the other QTL-Seq.