AbstractGenetic sexing strains (GSS) are an important tool in support of sterile insect technique (SIT) applications against insect pests and disease vectors. The yet unknowntemperature-sensitive lethal(tsl) gene and the recently identifiedwhite pupae(wp) gene have been used as selectable markers in the most successful GSS developed so far, theCeratitis capitata(medfly) VIENNA 8 GSS. The molecular identification of thetslgene may open the way for its use as a marker for the development of GSS in other insect pests and disease vectors of SIT importance. Prior studies have already shown that thetslgene is located on the right arm of chromosome 5, between thewpandZwloci (tslgenomic region). In the present study, we used genomic, transcriptomic, bioinformatic, and cytogenetic approaches to characterize and analyze this genomic region in wild-type andtslmutant medfly strains. Our results suggested the presence of 561 genes, with 322 of them carrying SNPs and/or insertion–deletion (indel) mutations in thetslgenomic region. Furthermore, comparative transcriptomic analysis indicated the presence of 32 differentially expressed genes, and bioinformatic analysis revealed the presence of 33 orthologs with a described heat-sensitive phenotype ofDrosophila melanogasterin this region. These data can be used in functional genetic studies to identify thetslgene(s) and the causal mutation(s) responsible for the temperature-sensitive lethal phenotype in medfly, and potentially additional genes causing a similar phenotype.