Alpha 1 chain (Colalpha1(I)) and alpha 2 chain (Colalpha2(I)) are universal components of type I collagen in tetrapods, but rainbow trout (Oncorhynchus mykiss) and zebrafish (Danio rerio) have a third: alpha 3 chain (Colalpha3(I)). This study tests whether Colalpha3(I) is a duplicate of Colalpha1(I) by whole-genome duplication (WGD) that occurred early in the ray-fin fish lineage. We also examine how their promoter sequence was modified after WGD. We cloned Colalpha1(I), Colalpha2(I) and Colalpha3(I) cDNAs and their promoters from flounder (Paralichthys olivaceus) and obtained corresponding sequences from the genome databanks of two pufferfishes Takifugu rubripes and Tetraodon nigroviridis, by BLAST-Search using flounder sequences. Phylogenetic analysis of N-terminal sequences of ca. 100 amino acids, including signal peptide and N-propeptide sequences before short triple helical domain, indicates that Colalpha3(I), found only in teleosts, is a duplicate of Colalpha1(1) by WGD. Colalpha1(I) and Colalpha3(I) genes begin to be transcribed at different stages of Takifugu embryogenesis, suggesting that their structure of promoter is modified differently after WGD. In flounder, Takifugu and Tetraodon, the structure of proximal region of promoter is highly conserved within Colalpha1(I) and within Colalpha3(I); no homology is apparent except for the TATA element motif between Colalpha1(I) and Colalpha3(I) of each species. Unexpectedly, zebrafish Colalpha1(I) promoter is more homologous to Colalpha3(I) of flounder and fugu than Colalpha1(I) is. These results suggest that each duplicated Colalpha1(I) gene promoter inherited a unique structure after WGD, but the manner of modification differed between the phylogenetically separated zebrafish and flounder/pufferfish lineages.