Sugar beets are attacked by several fungal pathogens that cause root damage, including Rhizoctonia solani, which causes the disease Rhizoctonia Crown and Root Rot (RCRR). Several Quantitative Trait Loci have been reported to harbor resistance genes to RCRR, but no RCRR resistance gene has been cloned from sugar beet or a crop wild relative. Discovering the identity of the genetic elements responsible for resistance will enable more precise molecular assisted breeding alongside a better understanding of the plant-pathogen interaction to drive future RCRR research efforts. In this study, we report a multifaceted genomics approach to map and characterize RCRR resistance from a highly resistant sugar beet prebreeding line. Utilizing bi-parental mapping, an RCRR susceptible parent line (03-124) was crossed with the highly resistant FC709-2 to produce F2 families. Multiple F2 families have been subjected to both field and greenhouse disease trials to study phenotypic segregation. Multiple genomic strategies, including Quantitative Trait Loci mapping using genotyping-by-sequencing and bulk segregant sequencing as well as genotyped using genotype by sequencing, were used to map the genomic loci harboring RCRR resistance in this population. To avoid reference genome bias, as well as provide a new tool for sugar beet pan-genomics and trait mapping, we developed the most complete and contiguous genome assembly for sugar beet to date from FC709-3, a more inbred and homogeneous derivative of FC709-2. Finally, we performed a comprehensive RNA expression profiling experiment using Illumina RNA sequencing to provide transcriptional support for genome annotation and to identify differentially expressed genes that are co-located in RCRR QTL in this mapping population. We anticipate that an enhanced understanding of the genetic sources of RCRR resistance will drive marker assisted selection in both pre-breeding and commercial breeding programs, as well as drive the discovery of novel RCRR resistance traits from crop wild relatives.