Due to the growing threat of emerging viral pathogens to global fruit production, understanding their molecular biology is essential for effective disease management. This chapter analyzes contemporary methodologies for molecular characterization and genome annotation of novel and re-emerging fruit viruses, including advanced sequencing technologies, such as short-read and long-read sequencing, which have improved the discovery and genomic profiling (genetic variants, mutations, genome structure, and expression patterns) of these pathogens. Plus, several molecular techniques for virus identification, genetic diversity, and viral population analysis, such as CRISPR-based assays and amplicon-based metagenomics, are covered. Principles and practical applications of de novo genome assembly and annotation pipelines are emphasized, incorporating artificial intelligence. Additionally, this manuscript includes approaches for the identification of open reading frames, predicting protein functions, mapping regulatory elements, and distinguishing evolutionary relationships through phylogenetic analyses. Furthermore, the chapter expands on complementary strategies used to validate genomic findings and refine structural and functional predictions by integrating genomic-transcriptomic evidence and combining computational tools with targeted experimental verification. Finally, this work also covers challenges researchers face during the annotation of complex viral genomes, showing endogenous viruses and diverse replication strategies examples, highlighting common sources of bioinformatic error, and proposing solutions to reduce misclassification or incomplete genome reconstruction. Summarizing, the overall objective was to provide a robust framework for high-quality genomic data generation, significant for developing accurate diagnostic tools, reporting on plant resistance improvement programs (breeding), and guiding sustainable control strategies to protect fruit crop health and support resilient agricultural systems capable of addressing emerging viral threats.