The mitochondrial cytochrome c oxidase subunit I ( COI ) gene is an effective molecular tool for the estimation of genetic variation and the identification of bird species. This molecular marker is used to differentiate among Chilean bird species by analyzing barcodes for 76 species (197 individuals), comprising 28 species with no previous barcode data and 48 species with sequences retrieved from the BOLD and GenBank databases. The DNA barcodes correctly identified 94.7% of the species analyzed (72 of 76 species). Mean intraspecific K2P distance was 0.3% (range 0–8.7%). Within the intraspecific divergence range, three species, Phrygilus gayi , Sephanoides sephanoides and Curaeus curaeus , showed relatively high intraspecific divergence (1.5–8.7%), possibly due to the presence of a species complex or geographic isolation of sub-populations. Mean interspecific K2P distance was 24.7% (range 1.3–43.5%). Consequently, the intraspecific K2P distance showed limited overlap with interspecific K2P distance. The mean intraspecific divergence in our study was similar to that found in temperate regions of South America (0.24%). However, it was approximately one order of magnitude lower than values reported for bird species in tropical regions of northern South America (1.8–2.13%). This result suggests that bird species from Chile show low levels of genetic structure and divergence. The small overlap between intra- and inter-specific distances implies that COI barcodes could be used as an effective tool to identify nearly all the Chilean bird species analyzed.