Multiple sequence alignment (MSA) may refer to the process or the result of sequence alignment of three or more biological sequences, generally protein, DNA, or RNA. In many cases, the input set of query sequences are assumed to have an evolutionary relationship by which they share a linkage and are descended from a common ancestor. From the resulting MSA, sequence homology can be inferred and phylogenetic analysis can be conducted to assess the sequences' shared evolutionary origins. Visual depictions of the alignment as in the image at right illustrate mutation events such as point mutations (single amino acid or nucleotide changes) that appear as differing characters in a single alignment column, and insertion or deletion mutations (indels or gaps) that appear as hyphens in one or more of the sequences in the alignment. Multiple sequence alignment is often used to assess sequence conservation of protein domains, tertiary and secondary structures, and even individual amino acids or nucleotides.

Computational algorithms are used to produce and analyse the MSAs due to the difficulty and intractability of manually processing the sequences given their biologically-relevant length. MSAs require more sophisticated methodologies than pairwise alignment because they are more computationally complex. Most multiple sequence alignment programs use heuristic methods rather than global optimization because identifying the optimal alignment between more than a few sequences of moderate length is prohibitively computationally expensive. On the other hand, heuristic methods generally fail to give guarantees on the solution quality, with heuristic solutions shown to be often far below the optimal solution on benchmark instances.