Exploring genetic diversity is essential for precise species delimitation, especially within taxonomically complex groups like passerine birds. Traditional morphological methods often fail to resolve species boundaries; however, DNA barcoding, particularly through the mitochondrial cytochrome c oxidase subunit I (COI) gene, provides a powerful complementary method for accurate species identification. This study establishes a comprehensive DNA barcode library for Iranian passerine birds, analyzing 546 COI sequences from 94 species across 23 families and 53 genera. There is a pronounced barcode gap, with average intraspecific divergence at 0.41% and interspecific divergence at 18.6%. Notable intraspecific variation emerged in the Persian nuthatch (Sitta tephronota) and the Lesser whitethroat (Curruca curruca), while the European goldfinch (Carduelis carduelis) and the grey-crowned goldfinch (Carduelis caniceps) showed limited genetic differentiation despite marked morphological distinctions. Phylogenetic analysis revealed significant east-west genetic splits in C. curruca and S. tephronota, reflecting Iran's geographic and zoogeographic boundaries. These findings demonstrate the effectiveness of DNA barcoding in elucidating biogeographic patterns, emphasizing Iran's key role as an ornithological crossroads for avian biodiversity. Moreover, our results suggest that much of the genetic variation in the COI gene arises from synonymous mutations, highlighting the role of purifying selection in shaping mtDNA diversity across species.