Nicotiana benthamiana, a native Australian species belonging to the agronomically valued Solanaceae family, is the species of choice for host-pathogen interactions and gene function analysis in plants. The popularity of N. benthamiana as a model plant system has seen exponential growth; however, its utilisation in forward genetics studies has been limited to date. Therefore, we are developing a platform for high-throughput trait mapping and gene discovery pipeline based on the recently assembled high-quality genomes of two phenotypically distinct N. benthamiana ecotypes, LAB and QLD. This will be used to investigate the contrasting response to anthocyanin production observed in the two N. benthamiana ecotypes. Transient over-expression of AcMYB110, an activation regulator of anthocyanin biosynthesis, enhances anthocyanin production in the wild QLD ecotype, but is detrimental in the LAB strain. Here, we utilised a cross-population between LAB and QLD ecotypes to investigate the segregation of these contrasting parental phenotypes in its progeny. The First Generation (F1) produced from the cross exclusively exhibited the purple QLD phenotype, and the progeny of F1 (F1S1) segregated in a 1:3 ratio for LAB and QLD phenotype, respectively, suggesting that the inability to accumulate anthocyanin in the N. benthamiana LAB strain is recessive in nature. We carried out bulk segregant analysis by utilising a pool of 40 individuals exhibiting the phenotype of LAB for whole genome sequencing followed by mapping of F1S1 reads to the respective parental genomes to identify the causal region of interest responsible for their phenotype.