To solve the current problem of inconsistent and inaccurate results of tissue marker gene identification in Drosophila melanogaster. Drosophila tissue marker genes were identified by Seurat as well as calculation of differential expression folds using transcriptomics data of seven shared tissues from Fly Cell Atlas and FlyAtlas2 databases, respectively. After cross-validation across datasets, literature comparison and gene function searches, accurate tissue marker genes were finally identified. From the two datasets, 575 and 5 202 tissue marker genes were identified, respectively, and 366 and 2 671 tissue marker genes were obtained after cross-validation across the datasets, and a total of 55 core tissue marker genes and 23 candidate tissue marker genes were obtained through literature comparison and gene function search in the FlyBase database. An accurate set of Drosophila tissue marker genes was established, providing an important reference for future Drosophila tissue type differentiation and cell type marker gene identification.