The tRNAscan-SE On-line resource provides an updated web server for detecting and analyzing transfer RNA (tRNA) genes. The new version, tRNAscan-SE 2.0, improves tRNA detection and functional prediction using advanced covariance models and integrates with the GtRNAdb database and UCSC genome browsers for enhanced biological context. The web server allows users to search for tRNA genes in their sequences and provides links to related genes in the GtRNAdb and genomic context in UCSC genome browsers. The new version also includes improved functional classification of tRNAs using isotype-specific covariance models, enabling more accurate identification of tRNA isotypes. The server supports various search modes, including a 'legacy' mode for backward compatibility. The tRNAscan-SE On-line web server can be accessed at http://trna.ucsc.edu/tRNAscan-SE/. The new version also includes a 'Infernal without HMM' search mode for low-scoring tRNA-like sequences. The tRNAscan-SE 2.0 software and search models produce slightly different bit scores compared to the previous version, but the relative rankings of previously detected tRNAs should remain largely unchanged. The server also provides a 'completeness' report for tRNA gene sets based on phylogenetic patterns observed across all genomes in the GtRNAdb. Future developments include metagenomic analysis of sequencing data containing an unknown mix of species and detection and classification of tRNA fragments in the context of full-length tRNA genes. The tRNAscan-SE web server is a valuable tool for researchers in the field of tRNA research.The tRNAscan-SE On-line resource provides an updated web server for detecting and analyzing transfer RNA (tRNA) genes. The new version, tRNAscan-SE 2.0, improves tRNA detection and functional prediction using advanced covariance models and integrates with the GtRNAdb database and UCSC genome browsers for enhanced biological context. The web server allows users to search for tRNA genes in their sequences and provides links to related genes in the GtRNAdb and genomic context in UCSC genome browsers. The new version also includes improved functional classification of tRNAs using isotype-specific covariance models, enabling more accurate identification of tRNA isotypes. The server supports various search modes, including a 'legacy' mode for backward compatibility. The tRNAscan-SE On-line web server can be accessed at http://trna.ucsc.edu/tRNAscan-SE/. The new version also includes a 'Infernal without HMM' search mode for low-scoring tRNA-like sequences. The tRNAscan-SE 2.0 software and search models produce slightly different bit scores compared to the previous version, but the relative rankings of previously detected tRNAs should remain largely unchanged. The server also provides a 'completeness' report for tRNA gene sets based on phylogenetic patterns observed across all genomes in the GtRNAdb. Future developments include metagenomic analysis of sequencing data containing an unknown mix of species and detection and classification of tRNA fragments in the context of full-length tRNA genes. The tRNAscan-SE web server is a valuable tool for researchers in the field of tRNA research.