The emergence and spread of Verticillium wilt were observed in cotton plants at the R.R. Foil Plant Science Research Center at Mississippi State during the late summer in 2013 and 2014. Several fungi with different morphology and growth characteristics were isolated from diseased cotton plants. Genomic DNA was extracted from the isolated fungal species and used in molecular typing via PCR amplification and DNA sequencing analysis of the ribosomal internal transcribed spacer (ITS) region. A total of five fungal genera were identified, and Verticillium sp. was the most frequently isolated genus. The isolated Verticillium strains could be Verticillium dahliae, Verticillium longisporum or even Verticillium albo-atrum. A PCR-based genotyping method using VTA2 (Verticillium transcription activator) gene specific primers confirmed that the isolated Verticillium strain was Verticillium dahliae, and it caused Verticillium wilt in Mississippi cotton plants. Pathogenicity tests (Koch’s postulates) confirmed the earlier qualitative identifications of Verticillium dahliae in the greenhouse.