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Aims: The main aim of this study was to fill a gap in barcoding data of the European Chironomidae of the Balkan region, developing and testing the efficiency of a reference DNA barcode library for ancient Lake Skadar basin species (Montenegro/Albania), a region of Europe never before subjected to barcoding studies on Chironomidae. Another aim was to test the efficiency of DNA barcoding for the identification of European Chironomidae, including the estimation of optimal identification thresholds, using >12,000 barcodes. Location: Lake Skadar basin and adjacent area (Montenegro/Albania). Methods: Through this study, 770 individuals of Chironomidae from the Lake Skadar region were barcoded, both at adult and pre‐imaginal stages. Adults were morphologically identified, while larvae were assigned to species by molecular identification, using different methods, of which the efficiency was tested, for a total of 97 different barcoded species. Results: The identification efficiency of the reference dataset developed for the Lake Skadar region was 98.6%, a value in line with that obtained when the identification efficiency for European Chironomidae was evaluated (95.8%), which confirms the accuracy of DNA barcoding for the identification of these insects. Moreover, we found that the optimal threshold for the molecular identification of the family is 1.6% nucleotide distance, though more specific thresholds are suggested for the identification of species belonging to Chironomidae subfamilies, since they are related to lower identification errors than to the use of a general threshold. The analysis of inconsistency between molecular and morphological identification shed light on taxonomic issues within European Chironomidae. Previously postulated species synonyms were confirmed, and also further cases requiring deeper investigation were detected. Main conclusions: Our de novo DNA barcode library was shown to have a high identification efficiency. Taxon‐specific thresholds increase the efficacy of molecular identification. Hypothesized species synonyms could be validated through molecular techniques. [ABSTRACT FROM AUTHOR]