ABSTRACT Anthracnose diseases caused by Colletotrichum species are among the most common fungal diseases. These symptoms typically manifest as dark, sunken lesions on leaves, stems, and fruit. In China, mango anthracnose seriously affects fruit yield and quality. Genome sequencing of several species shows the presence of mini-chromosomes. These are thought to contribute to virulence, but their formation and activity remain to be fully elucidated. Here, we assembled 17 Colletotrichum genomes (16 isolated from mango plus one from persimmon) through PacBio long-read sequencing. Half of the assembled scaffolds had telomeric repeats at both ends indicating full-length chromosomes. Based on comparative genomics analysis at interspecies and intraspecies levels, we identified extensive chromosomal rearrangements events. We analyzed mini-chromosomes of Colletotrichum spp. and found large variation among close relatives. In C. fructicola , homology between core chromosomes and mini-chromosomes suggested that some mini-chromosomes were generated by recombination of core chromosomes. In C. musae GZ23-3, we found 26 horizontally transferred genes arranged in clusters on mini-chromosomes. In C. asianum FJ11-1, several potential pathogenesis-related genes on mini-chromosomes were upregulated, especially in strains with highly pathogenic phenotypes. Mutants of these upregulated genes showed obvious defects in virulence. Our findings provide insights into the evolution and potential relationships to virulence associated with mini-chromosomes. IMPORTANCE Colletotrichum is a cosmopolitan fungal genus that seriously affects fruit yield and quality of many plant species. Mini-chromosomes have been found to be related to virulence in Colletotrichum . Further examination of mini-chromosomes can help us elucidate some pathogenic mechanisms of Colletotrichum . In this study, we generated novel assemblies of several Colletotrichum strains. Comparative genomic analyses within and between Colletotrichum species were conducted. We then identified mini-chromosomes in our sequenced strains systematically. The characteristics and generation of mini-chromosomes were investigated. Transcriptome analysis and gene knockout revealed pathogenesis-related genes located on mini-chromosomes of C. asianum FJ11-1. This study represents the most comprehensive investigation of chromosome evolution and potential pathogenicity of mini-chromosomes in the Colletotrichum genus.