The article introduces the updated tRNAscan-SE On-line resource, which integrates advanced tRNA gene detection and functional prediction with enhanced biological context. The new version, tRNAscan-SE 2.0, uses Infernal 1.1 software for improved covariance model search, updated models for broader tRNA diversity, and better functional classification using isotype-specific models. The web server now provides dynamic links to the Genomic tRNA Database (GtRNAb) and UCSC genome browsers, allowing users to explore tRNA predictions in their genomic context. The article also discusses the development of isotype-specific models to identify potential chimeric tRNAs and future plans for metagenomic analysis and fragment detection. The tRNAscan-SE On-line resource is accessible at http://trna.ucsc.edu/tRNAscan-SE/.The article introduces the updated tRNAscan-SE On-line resource, which integrates advanced tRNA gene detection and functional prediction with enhanced biological context. The new version, tRNAscan-SE 2.0, uses Infernal 1.1 software for improved covariance model search, updated models for broader tRNA diversity, and better functional classification using isotype-specific models. The web server now provides dynamic links to the Genomic tRNA Database (GtRNAb) and UCSC genome browsers, allowing users to explore tRNA predictions in their genomic context. The article also discusses the development of isotype-specific models to identify potential chimeric tRNAs and future plans for metagenomic analysis and fragment detection. The tRNAscan-SE On-line resource is accessible at http://trna.ucsc.edu/tRNAscan-SE/.