ABSTRACT. The avian family Ardeidae comprises long-legged freshwater and coastal birds. There has been considerable disagreement concerning the intrafamilial relationships of Ardeidae. Mitochondrial cytochrome c oxidase subunit I (COI) was used as a marker for the identification and phylogenetic analysis of avian species. In the present study, we analyzed the COI barcodes of 32 species from 17 genera belonging to the family Ardeidae. Each bird species possessed a barcode distinct from that of other bird species except for Egretta thula and E. garzetta, which shared one barcoding sequence. Kimura two-parameter distances were calculated between barcodes. The average genetic distance between species was 34-fold higher than the average genetic distance within species. Neighbor-joining and maximum likelihood methods were used to construct phylogenetic trees. Most species could be discriminated by their distinct clades in the phylogenetic tree. Both methods of phylogenetic reconstruction suggested that Zebrilus, Tigrisoma, and Cochlearius were an offshoot of the primitive herons. COI gene analysis suggested that the other herons could be divided