MUSCLE is a multiple sequence alignment (MSA) program that improves upon previous methods by offering faster and more accurate alignment. It is designed to handle large datasets efficiently and provides several options, including MUSCLE-fast for high-throughput applications. The algorithm uses a combination of progressive alignment and iterative refinement to improve alignment accuracy. It employs a similarity measure based on fractional identity and k-mer counting, and uses a distance matrix to construct a tree for alignment. The tree is then used to build a progressive alignment, which is refined through iterative steps. MUSCLE-fast is the fastest variant, achieving high accuracy in times that are typically two to three orders of magnitude less than other programs. It can align 1,000 sequences of average length 282 in 21 seconds on a desktop computer. MUSCLE offers a range of options for improved speed and accuracy compared to existing programs and is freely available at http://www.drive5.com/muscle.MUSCLE is a multiple sequence alignment (MSA) program that improves upon previous methods by offering faster and more accurate alignment. It is designed to handle large datasets efficiently and provides several options, including MUSCLE-fast for high-throughput applications. The algorithm uses a combination of progressive alignment and iterative refinement to improve alignment accuracy. It employs a similarity measure based on fractional identity and k-mer counting, and uses a distance matrix to construct a tree for alignment. The tree is then used to build a progressive alignment, which is refined through iterative steps. MUSCLE-fast is the fastest variant, achieving high accuracy in times that are typically two to three orders of magnitude less than other programs. It can align 1,000 sequences of average length 282 in 21 seconds on a desktop computer. MUSCLE offers a range of options for improved speed and accuracy compared to existing programs and is freely available at http://www.drive5.com/muscle.