Verticillium dahliae(V. dahliae) is a notorious soil-borne pathogen causing Verticillium wilt in more than 400 dicotyledonous plants, including a wide range of economically important crops, such as cotton, tomato, lettuce, potato, and romaine lettuce, which can result in extensive economic losses. In the last decade, several studies have been conducted on the physiological and molecular mechanisms of plant resistance toV. dahliae. However, the lack of a complete genome sequence with a high-quality assembly and complete genomic annotations forV. dahliaehas limited these studies. In this study, we produced a full genomic assembly forV. dahliaeVD991 using Nanopore sequencing technology, consisting of 35.77 Mb across eight pseudochromosomes and with a GC content of 53.41%. Analysis of the genome completeness assessment (BUSCO alignment: 98.62%; Illumina reads alignment: 99.17%) indicated that our efforts resulted in a nearly complete and high-quality genomic assembly. We selected 25 species closely related toV. dahliaefor evolutionary analysis, confirming the evolutionary relationship betweenV. dahliaeand related species, and the identification of a possible whole genome duplication event inV. dahliae. The interaction between cotton andV. dahliaewas investigated by transcriptome sequencing resulting in the identification of many genes and pathways associated with cotton disease resistance andV. dahliaepathogenesis. These results will provide new insights into the pathogenic mechanisms ofV. dahliaeand contribute to the cultivation of cotton varieties resistant to Verticillium wilt.