TranslatorX is a web server that aligns protein-coding nucleotide sequences based on their corresponding amino acid translations. It addresses the challenge of aligning nucleotide sequences, which are more prone to sequence similarity degradation compared to amino acids. The server offers several features, including the use of multiple genetic codes, a variety of multiple alignment programs, translation of ambiguous codons, and an innovative method to clean nucleotide alignments using GBlocks based on protein information. It also provides rich output, including graphical visualization of alignments, codon-based alignments colored according to amino acids, and measures of compositional bias. The server is designed to improve the accuracy of nucleotide sequence alignments by translating DNA sequences into amino acids, aligning these, and then back-translating the alignment to nucleotides. This approach is more effective than direct nucleotide alignment, especially in regions with high substitution rates and frequent indels. TranslatorX allows users to assign different genetic codes to each sequence and offers a range of alignment programs, including Muscle, Mafft, T-Coffee, Prank, and ClustalW. The server also provides an innovative method for cleaning nucleotide alignments by using GBlocks on the amino acid alignment to remove ambiguous regions. This results in more accurate alignments with higher confidence in positional homology. The output includes alignments visualized in Jalview, which allows for further analysis and refinement. In a comparison of TranslatorX with direct nucleotide alignments, the results showed that back-translated alignments produced more accurate phylogenetic trees, with higher support for good nodes and lower support for bad nodes. The use of amino acid information improved the alignment performance, especially in variable regions. Additionally, the compositional bias in the nucleotide sequences was reduced after alignment cleaning, indicating that the cleaning process helped in minimizing systematic errors in phylogenetic analyses. The study highlights the importance of considering amino acid information when aligning nucleotide sequences, as it leads to more accurate phylogenetic reconstructions. The results suggest that using amino acid alignments can maximize the correct interpretation of positional homology in variable regions, resulting in better phylogenetic performance. TranslatorX is a valuable tool for researchers aiming to improve the accuracy of nucleotide sequence alignments and subsequent phylogenetic analyses.TranslatorX is a web server that aligns protein-coding nucleotide sequences based on their corresponding amino acid translations. It addresses the challenge of aligning nucleotide sequences, which are more prone to sequence similarity degradation compared to amino acids. The server offers several features, including the use of multiple genetic codes, a variety of multiple alignment programs, translation of ambiguous codons, and an innovative method to clean nucleotide alignments using GBlocks based on protein information. It also provides rich output, including graphical visualization of alignments, codon-based alignments colored according to amino acids, and measures of compositional bias. The server is designed to improve the accuracy of nucleotide sequence alignments by translating DNA sequences into amino acids, aligning these, and then back-translating the alignment to nucleotides. This approach is more effective than direct nucleotide alignment, especially in regions with high substitution rates and frequent indels. TranslatorX allows users to assign different genetic codes to each sequence and offers a range of alignment programs, including Muscle, Mafft, T-Coffee, Prank, and ClustalW. The server also provides an innovative method for cleaning nucleotide alignments by using GBlocks on the amino acid alignment to remove ambiguous regions. This results in more accurate alignments with higher confidence in positional homology. The output includes alignments visualized in Jalview, which allows for further analysis and refinement. In a comparison of TranslatorX with direct nucleotide alignments, the results showed that back-translated alignments produced more accurate phylogenetic trees, with higher support for good nodes and lower support for bad nodes. The use of amino acid information improved the alignment performance, especially in variable regions. Additionally, the compositional bias in the nucleotide sequences was reduced after alignment cleaning, indicating that the cleaning process helped in minimizing systematic errors in phylogenetic analyses. The study highlights the importance of considering amino acid information when aligning nucleotide sequences, as it leads to more accurate phylogenetic reconstructions. The results suggest that using amino acid alignments can maximize the correct interpretation of positional homology in variable regions, resulting in better phylogenetic performance. TranslatorX is a valuable tool for researchers aiming to improve the accuracy of nucleotide sequence alignments and subsequent phylogenetic analyses.