The data resource contains Drosophila simulans backcross female mate choice (phenotype) aligned with whole-genome genotypes of 784 female backcross progeny. The data were generated in a laboratory at Stony Brook University, New York and each test was conducted in 28.5 x 95mm plastic vials. Phenotype data was collected in 2015 and the final analysis was completed in 2021. Illumina libraries were sequenced at University of Oregon G3 Genomics Center, and the multiplexed shotgun genotyping (MSG) software was run at Janelia Research Campus, Virginia. The experiments were conducted to align the genotype of females with behavioural phenotype, female preference. Three test females were allowed to choose from three males of either species. ‘Choice’ was determined by copulation, and the species of ‘chosen’ male was recorded before gDNA was extracted from the female. Standard molecular biology was used to generate Illumina libraries from individual backcross females for sequencing. Deniz Erezyilmaz was responsible for data collection and interpretation. Those genomes with incomplete or inconsistent genomes due to sparse reads/coverage were eliminated from the analysis. The research was funded through NERC grant NE/S010351/1. Investigating the dual role of mate choice genes in behavioural isolation and hybridization. Full details about this nonGeographicDataset can be found at https://doi.org/10.5285/8dde3529-1cf7-4b0f-907d-a1631f38afd7