[Objective] Cytospora canker diseases are among the most important forest diseases, causing devastating economic losses and ecological damage. To understand the genome structure and genetic variation of Cytospora species with different host range, we performed a high-quality genome sequencing of Cytospora piceae and the corresponding comparative genomic analysis. Our study will provide a stepping stone to indicating the molecular mechanism of the interaction between Cytospora spp. and their hosts, and controlling the Cytospora canker disease.[Methods] The draft genome of Cytospora piceae was sequenced by PacBio sequencing technology, and was annotated. The genomic variation, host range determinants and the unique virulence-related gene families of the four Cytospora species were analyzed through comparative genomics tools.[Results] The assembled genome size of C. piceae was 39.25 Mb, with a GC content of 51.79%. The phylogenetic tree based on single-copy orthologue genes showed that C. piceae was closely related to Cytospora chrysosperma, and Valsa mali and Valsa pyri was in the same clade. GC content distribution analysis indicated repeat-induced point mutation activity in all Cytospora species, and C. piceae had the strongest repeat-induced point mutation activity. The carbohydrate active enzymes of all four Cytospora spp. was similar in number. Among plant cell wall degrading enzymes, the auxiliary activity family 3 and 7 related to lignin degradation expanded significantly, while the auxiliary activity family 5, the key enzymes for lignin degradation, was absent in Cytospora spp.. The number of genes in the glycoside hydrolase family 28 and 8 of key enzymes for pectin degradation in the C. piceae and C. chrysosperma genome was similar to that of V. mali and V. pyri. C. piceae and other three Cytospora species all had more major facilitator superfamily transporters and fewer ATP-binding cassette family transporters. In addition, C. piceae contained more Drug:H⁺ Antiporter-2, Pleiotropic Drug Resistance and Multidrug Resistance Exporter transporters, while V. mali contained less Drug:H⁺ Antiporter-1 transporters. Gene Ontology functional classification indicated that the genes of all Cytospora species concentrated on hydrolase activity, V. mali has the highest number of the genes of this class, and the biological processes were mainly related to carbohydrate metabolism, pectin catabolic and oxidation reduction processes. Among the secondary metabolism core genes, C. piceae had fewer polyketide synthases genes than V. mali and V. pyri. Among the four C. piceae specific secondary metabolites genes, three were nonribosomal peptide synthases genes.[Conclusion]] The carbohydrate active enzymes of four Cytospora species were similar in number, which showed strong pectin degradation ability. A complex pattern of presence or absence of nonribosomal peptide synthases genes in the secondary metabolites core genes, and the expanded multidrug transporters of four Cytospora species were observed, which indicated that they are likely to play an important role in host selection of Cytospora species.