ARWEN is a program designed to detect tRNA genes in metazoan mitochondrial nucleotide sequences. It achieves near 100% detection of previously annotated tRNA genes. The program uses a heuristic algorithm to identify tRNA genes by searching for specific structural features, including hairpin structures with specific stem and loop lengths. It considers various structural and sequence features, such as base pairing interactions, tertiary structure interactions, and consensus sequence motifs, to assign scores to candidate sequences. The program also accounts for differences in genetic code used by mitochondria and adjusts for variations in GC content. ARWEN was tested against multiple datasets, including mammalian mitochondrial tRNA gene sequences from the Mamit-tRNA database and metazoan mitochondrial tRNA gene sequences from the OGRe database. It achieved high detection sensitivity compared to tRNAscan-SE, with a detection rate of 99.4% for a set of 23 mitochondrial genomes containing 469 tRNAs. ARWEN also ran significantly faster than tRNAscan-SE, completing scanning the 23 genomes almost 30 times faster. However, ARWEN had a higher number of false positives than tRNAscan-SE. When the threshold values were adjusted to match tRNAscan-SE's selectivity, ARWEN's sensitivity dropped to 81.7%, slightly higher than tRNAscan-SE's 72.1%. ARWEN is implemented in C and can be downloaded from its website. It allows users to upload sequences and run the program on a server. The program can handle sequences up to 2 MB in length and provides detailed output, including secondary structure, anticodon position, and amino acid iso-acceptor species for each candidate tRNA. ARWEN is more sensitive than tRNAscan-SE in detecting tRNA genes but has a higher rate of false positives. However, when used together, the two programs can improve the accuracy of tRNA annotation in metazoan mitochondrial sequences. ARWEN is particularly effective for mammalian mitochondrial genomes, where it detects nearly all tRNA genes, while tRNAscan-SE often misses many. ARWEN's results suggest that it is more suitable for detecting tRNA genes in metazoan mitochondrial sequences than tRNAscan-SE.ARWEN is a program designed to detect tRNA genes in metazoan mitochondrial nucleotide sequences. It achieves near 100% detection of previously annotated tRNA genes. The program uses a heuristic algorithm to identify tRNA genes by searching for specific structural features, including hairpin structures with specific stem and loop lengths. It considers various structural and sequence features, such as base pairing interactions, tertiary structure interactions, and consensus sequence motifs, to assign scores to candidate sequences. The program also accounts for differences in genetic code used by mitochondria and adjusts for variations in GC content. ARWEN was tested against multiple datasets, including mammalian mitochondrial tRNA gene sequences from the Mamit-tRNA database and metazoan mitochondrial tRNA gene sequences from the OGRe database. It achieved high detection sensitivity compared to tRNAscan-SE, with a detection rate of 99.4% for a set of 23 mitochondrial genomes containing 469 tRNAs. ARWEN also ran significantly faster than tRNAscan-SE, completing scanning the 23 genomes almost 30 times faster. However, ARWEN had a higher number of false positives than tRNAscan-SE. When the threshold values were adjusted to match tRNAscan-SE's selectivity, ARWEN's sensitivity dropped to 81.7%, slightly higher than tRNAscan-SE's 72.1%. ARWEN is implemented in C and can be downloaded from its website. It allows users to upload sequences and run the program on a server. The program can handle sequences up to 2 MB in length and provides detailed output, including secondary structure, anticodon position, and amino acid iso-acceptor species for each candidate tRNA. ARWEN is more sensitive than tRNAscan-SE in detecting tRNA genes but has a higher rate of false positives. However, when used together, the two programs can improve the accuracy of tRNA annotation in metazoan mitochondrial sequences. ARWEN is particularly effective for mammalian mitochondrial genomes, where it detects nearly all tRNA genes, while tRNAscan-SE often misses many. ARWEN's results suggest that it is more suitable for detecting tRNA genes in metazoan mitochondrial sequences than tRNAscan-SE.