Grapevine trunk diseases (GTD) are widely distributed across grape producing regions around the world. These diseases are responsible for large reductions in productivity and longevity of the vineyards. A diverse group of fungi commonly known as Grapevine trunk pathogens (GTP) are the main causing agents of these diseases. Pathogenic species of the genus Botryosphaeria, Neofusicoccum, Lasiodiplodia, Diplodia, Eutypa, Phaeoacremonium, Phaeomoniella and others are among the main causal agents. Additionally, some Basidiomycetes a have also been associated with certain. The objective of this dissertation was to contribute to the understanding of the virulence and evolution of the GTPs through comparative genomics and pangenomics of different sets of grapevine trunk pathogens. In the first chapter, high-quality phased genome assemblies of four basidiomycete species associated with esca disease complex were produced. The genomic and functional comparisons identified potential virulence factors, suggesting their roles in disease development. Similar to other white-rot fungi, these four genomes encoded a variety of lignin peroxidases and carbohydrate-active enzymes (CAZymes). The analysis of gene family expansion and contraction revealed dynamic evolutionary patterns, particularly in genes related to secondary metabolite production, plant cell wall decomposition, and xenobiotic degradation. In the second chapter, a family level comparison of the Botryosphaeriaceae family was made. Measures of in planta virulence and phylogenomic comparisons of seventeen species known as trunk pathogens revealed significant variation in terms of the length of the internal wood lesions caused by the species and genus-specific expansion of gene families representing putative virulence factors involved in toxin production and mobilization, wood degradation, and nutrient uptake. In the third chapter, a pangenomic approach was followed. Fifty isolates from viticulture regions worldwide were sequenced and assembled into reference-free pangenomes of Eutypa lata, Neofusicoccum parvum, and Phaeoacremonium minimum. Examination of genomic diversity and pangenome structure revealed contrasting distributions of putative virulence factors in the core, dispensable, and private genomes of each pangenome. Transposable elements and virulence factors under positive selection were enriched in the dispensable regions of the pangenomes. In the fourth chapter, a metabarcoding analysis of grapevine trunk-associated Ascomycetes was performed on Californian grapevine nursery material. Mother plants, propagated material, and abiotic environmental samples were analyzed. By optimizing the taxonomy assignment protocol, beneficial and pathogenic species were identified in all the materials analyzed. The results show that fungal diversity was highly impacted by the nursery where the material came from and the stage of the propagation process.