Mutational change in a codon that results in a change in the specified amino acid.

Nonsynonymous substitutions definitively are not silent mutations since they result in change in . Nonsynonymous substitutions therefore have some reasonable likelihood of impacting an organism's physiology and therefore fitness.

A nonsynonymous substitution to a first approximation is assumed to be less likely to represent a neutral mutation. than an otherwise equivalent synonymous substitution. The fixation of such a mutation therefore is presumed to be more likely a consequence of natural selection than the fixation of a synonymous substitution. These ideas are directly applicable to the concept of dN/dS.

Note that nonsynonymous substitutions are considered to vary in the likely intensity of their modification of protein structure and therefore their potential impact on protein functioning. This impact can to a degree be predicted based on similarities between the affected amino acid and its replacement, that is, in terms of the size, shape, and chemistry of the two amino acids, with similar size, shape, and chemistry resulting in potentially more conservative rather than radical change in resulting protein structure.