The dataset contains 412 records of Diptera species from the families Limoniidae, Pediciidae and Tipulidae, collected between 2003 to 2019 in Portugal, including the Azores and Madeira archipelagos (Ferreira et al., 2021; Oosterbroek et al., 2020; Starý, 2014). Specimens have been identified to species or subspecies level, in a total of 83 species representing 58% of the Craneflies known for Portugal. Specimens were captured during fieldwork directed specifically for the sampling of Diptera using different methodologies and the majority was stored in 96% ethanol. 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 the EnvMetaGen and PORBIOTA projects. The dataset “DNA barcodes of Portuguese Diptera 02 - Limoniidae, Pediciidae and Tipulidae” is part of a group of Diptera datasets published by IBI, the first of which, “The InBIO Barcoding Initiative Database: Diptera 01” (https://doi.org/10.15468/q1bvt3) has already been made available through GBIF. DNA barcode sequences were deposited in BOLD (Barcode of Life Data System) and GenBank databases. All DNA extracts are deposited in the IBI collection at the CIBIO (Research Center in Biodiversity and Genetic Resources).