A nonsynonymous substitution is a nucleotide mutation that alters the amino acid sequence of a protein. Nonsynonymous substitutions differ from synonymous substitutions, which do not alter amino acid sequences and are (sometimes) silent mutations. As nonsynonymous substitutions result in a biological change in the organism, they are subject to natural selection.
Nonsynonymous substitutions at a certain locus can be compared to the synonymous substitutions at the same locus to obtain the K/K ratio. This ratio is used to measure the evolutionary rate of gene sequences. If a gene has lower levels of nonsynonymous than synonymous nucleotide substitution, then it can be inferred to be functional because a K/K ratio < 1 is a hallmark of sequences that are being constrained to code for proteins.
 Nonsynonymous substitutions are also referred to as replacement mutations.