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    The dataset contains 203 records of Diptera species collected from 2014 to 2018 in continental Portugal (Ferreira et al., 2020). The species represented in the dataset, 154 in total, correspond to about 10% of the known fly diversity of continental Portugal, and contribute to the knowledge on the DNA barcodes and distribution of Portuguese Diptera. Specimens were captured during fieldwork directed specifically for the sampling of Diptera using different methodologies and stored in 96% ethanol. All specimens were morphologically identified to species level. A tissue sample, usually a leg, was collected from each individual, from which DNA was extracted. The DNA barcoding of these specimens was conducted within the InBIO Barcoding Initiative (IBI), funded by EnvMetaGen and PORBIOTA projects. DNA barcode sequences were deposited in BOLD (Barcode of Life Data System) online database. Preserved specimens and DNA extracts are deposited in the IBI collection at the CIBIO (Research Center in Biodiversity and Genetic Resources).