KOBAS 2.0 is a web server designed to annotate and identify enriched pathways and diseases based on input sets of genes. It integrates knowledge from 1327 species across 5 pathway databases (KEGG PATHWAY, PID, BioCyc, Reactome, and Panther) and 5 human disease databases (OMIM, KEGG DISEASE, FunDO, GAD, and NHGRI GWAS Catalog). KOBAS 2.0 supports both ID mapping and cross-species sequence similarity mapping, allowing for the annotation of genes from species not well-represented in existing pathway databases. The tool performs statistical tests to identify significantly enriched pathways and diseases, using methods such as binomial, chi-square, Fisher’s exact, and hypergeometric tests, with FDR correction. KOBAS 2.0 is available at http://kobas.cbi.pku.edu.cn and can be accessed through a standalone command-line program or a user-friendly web interface. The tool has been shown to provide more specific and informative results compared to other pathway analysis tools, particularly when using sequence similarity mapping.KOBAS 2.0 is a web server designed to annotate and identify enriched pathways and diseases based on input sets of genes. It integrates knowledge from 1327 species across 5 pathway databases (KEGG PATHWAY, PID, BioCyc, Reactome, and Panther) and 5 human disease databases (OMIM, KEGG DISEASE, FunDO, GAD, and NHGRI GWAS Catalog). KOBAS 2.0 supports both ID mapping and cross-species sequence similarity mapping, allowing for the annotation of genes from species not well-represented in existing pathway databases. The tool performs statistical tests to identify significantly enriched pathways and diseases, using methods such as binomial, chi-square, Fisher’s exact, and hypergeometric tests, with FDR correction. KOBAS 2.0 is available at http://kobas.cbi.pku.edu.cn and can be accessed through a standalone command-line program or a user-friendly web interface. The tool has been shown to provide more specific and informative results compared to other pathway analysis tools, particularly when using sequence similarity mapping.