This protocol describes how to build phylogenetic trees from molecular data using MEGA, a comprehensive program that simplifies the process by integrating all steps into a single interface. Phylogenetic analysis is often perceived as complex, but MEGA makes it accessible for novices. The process involves four main steps: (1) identifying and acquiring homologous sequences, (2) aligning the sequences, (3) estimating the tree, and (4) presenting the tree. Step 1 involves using MEGA's built-in BLAST search to find homologous sequences from databases like NCBI. Users can choose between different BLAST algorithms depending on the similarity of the sequences. Once sequences are identified, they are added to the alignment. Step 2 involves aligning the sequences using either ClustalW or MUSCLE. MUSCLE is generally preferred for its accuracy. Alignment can be done directly within MEGA or using external programs like Guidance for more advanced alignment options. Step 3 involves estimating the phylogenetic tree using maximum likelihood (ML) methods. MEGA automatically selects the best substitution model for the data. The ML tree is then constructed, and the reliability of the tree can be assessed using the bootstrap method. Step 4 involves presenting the tree in a format suitable for publication. MEGA offers various tree formats, including unrooted and rooted trees. The tree should be saved in a graphics file format like PDF for publication. It is important to specify whether the tree is unrooted or rooted in the figure legend to avoid misinterpretation. MEGA is available for both Windows and Mac computers and provides a user-friendly interface for all steps of phylogenetic analysis. Alternative programs like PhyML and SeaView can also be used for tree estimation and drawing. The protocol emphasizes the importance of selecting appropriate sequences, accurate alignment, and proper tree presentation to ensure the reliability and clarity of the phylogenetic analysis.This protocol describes how to build phylogenetic trees from molecular data using MEGA, a comprehensive program that simplifies the process by integrating all steps into a single interface. Phylogenetic analysis is often perceived as complex, but MEGA makes it accessible for novices. The process involves four main steps: (1) identifying and acquiring homologous sequences, (2) aligning the sequences, (3) estimating the tree, and (4) presenting the tree. Step 1 involves using MEGA's built-in BLAST search to find homologous sequences from databases like NCBI. Users can choose between different BLAST algorithms depending on the similarity of the sequences. Once sequences are identified, they are added to the alignment. Step 2 involves aligning the sequences using either ClustalW or MUSCLE. MUSCLE is generally preferred for its accuracy. Alignment can be done directly within MEGA or using external programs like Guidance for more advanced alignment options. Step 3 involves estimating the phylogenetic tree using maximum likelihood (ML) methods. MEGA automatically selects the best substitution model for the data. The ML tree is then constructed, and the reliability of the tree can be assessed using the bootstrap method. Step 4 involves presenting the tree in a format suitable for publication. MEGA offers various tree formats, including unrooted and rooted trees. The tree should be saved in a graphics file format like PDF for publication. It is important to specify whether the tree is unrooted or rooted in the figure legend to avoid misinterpretation. MEGA is available for both Windows and Mac computers and provides a user-friendly interface for all steps of phylogenetic analysis. Alternative programs like PhyML and SeaView can also be used for tree estimation and drawing. The protocol emphasizes the importance of selecting appropriate sequences, accurate alignment, and proper tree presentation to ensure the reliability and clarity of the phylogenetic analysis.