Introduction: Tephritidae, the true fruit flies, is a family of Diptera including major agricultural pests threatening horticultural production worldwide. Within the framework of the EU H2020 FF-IPM project, we provided reference DNA barcodes to develop or expand the existing reference libraries for two main agricultural pests, Bactrocera dorsalis and Ceratitis capitata, and related taxa within genera of the subfamily Dacinae. Unlike the classical approach of generating DNA barcodes through Sanger Sequencing, we bioinformatically extracted COI DNA barcodes from Whole Genome Sequencing (WGS) data at genome coverage 10x. Methods: In this respect, 150bp paired-end reads were generated. The raw genomic reads were filtered using fastp and mapped to a related mitochondrial reference genome like C. capitata (NC_000857.1) or B. dorsalis (NC_008748.1) through bwa mem. The resulting read mappings were processed with samtools consensus to call variants. A multiple sequence alignment was made with MAFFT, and the COI sequences were extracted based on the annotation of the related mitochondrial reference genome. Results: DNA barcodes were generated from more than 1000 specimens consisting of more than 50 different species and made available through the open-access platforms of BOLD (Barcode of Life Data System) and GENBANK . Conclusion: These data expand the geographic coverage and temporal sampling of Tephritidae in the open-access repositories of COI DNA barcodes and contributes to the DNA barcoding identification of widespread agricultural pests. The bioinformatically recovered DNA barcodes can be linked to the corresponding WGS data available on the online platform of the Royal Museum for Central Africa.