The utilization of three important freshwater fish skins including tra catfish (Pangasianodon hypophthalmus), clown knifefish (Chitala ornata), and tilapia (Oreochromis niloticus) in Mekong Delta Region, Vietnam for acid-soluble collagen (ASC) extraction was investigated. From the SDS-PAGE profile, ASCs can be identified as type I collagen (consisting of two different α- chains and β component in protein pattern).The solubility among individual collagens was highest at pH 1–2, depending on fish species. FTIR spectra showed a close relationship between the lowest wavelength number in amide I and amide III regions and the stabilization of the triple-helical structure in ASC from clown knifefish. ASC from clown knifefish skin had highest ASC yield and contained highest amino acid content as well as the highest denaturation temperature (33.09°C) in comparison with ASCs from tra catfish and tilapia skin.