• RUS is a major dominant gene controlling quantitative resistance, inherited from Populus trichocarpa, whereas R1 is a gene governing qualitative resistance, inherited from P. deltoides. • Here, we report a reiterative process of concomitant fine-scale genetic and physical mapping guided by the P. trichocarpa genome sequence. The high-resolution linkage maps were developed using a P. deltoides × P. trichocarpa progeny of 1415 individuals. RUS and R1 were mapped in a peritelomeric region of chromosome 19. Markers closely linked to RUS were used to screen a bacterial artificial chromosome (BAC) library constructed from the P. trichocarpa parent, heterozygous at the locus RUS. • Two local physical maps were developed, one encompassing the RUS allele and the other spanning rUS. The alignment of the two haplophysical maps showed structural differences between haplotypes. The genetic and physical maps were anchored to the genome sequence, revealing genome sequence misassembly. Finally, the RUS locus was localized within a 0.8-cM interval, whereas R1 was localized upstream of RUS within a 1.1-cM interval. • The alignment of the genetic and physical maps with the local reorder of the chromosome 19 sequence indicated that RUS and R1 belonged to a genomic region rich in nucleotide-binding site leucine-rich repeat (NBS-LRR) and serine threonine kinase (STK) genes.