Fishes living in Arctic and Antarctic regions are adapted to the freezing seawater temperatures, which ostensibly due to adaptive modification of the sequence and structure of their biological molecules enabling their functioning in extreme cold. In this study, we investigate the substitution patterns of nucleotides and amino acids in the coding DNA sequences between three tropical fishes and two Antarctic fishes and two Arctic fishes. We want to characterize the preferences Polar fishes show preference for GCs in both synonymous and nonsynonymous substitution when compared with tropical fishes. GC bias in nonsynonymous substitution is linked with small amino acid preference in polar fishes. Investigation on amino acid substitution shows that polar fishes prefer small amino acids, and that the substitution pattern of Arctic fishes are more biased than that of Antarctic fishes when compared with tropical fishes. We predicted the secondary structure of the protein sequences and investigated the secondary structure substitution pattern. We found that frequency of the coils is increased in polar fishes when compared with tropical fishes, which lead to increase of structural flexibility of the proteins in polar fishes. This study indicates that increased GC content of the coding DNA sequences leads to the increase of small amino acids and consequently increases the flexibility of proteins in polar fishes, which may be an important cold-adaptation mechanism for polar fishes.