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The United States is the third largest producer of cotton and the largest exporter of cotton globally. Fusarium wilt, caused by the soilborne fungal pathogen Fusarium oxysporum f. sp. vasinfectum (Fov), was estimated to cause a $21 million cotton yield loss in 2022. Historically, Alabama was an important producer of cotton in the Southeastern United States and was the first state in which Fusarium wilt on cotton was described. To assess the genetic diversity of Fov field isolates in Alabama, 118 field isolates were collected from six counties across the state from 2014 to 2016. Phylogenetic analysis using TEF1 and RPB2 placed the Fov field isolates into 18 haplotypes. Upon profiling the Tfol transposon insertion in the NAT gene, it was determined that no race 4 isolates were recovered in Alabama. Representatives of all field isolate haplotypes caused disease on Upland cotton variety Rowden in a hydroponic test tube assay. Two haplotype A isolates were the most aggressive isolates recovered, and haplotype A isolate TF1 was more aggressive than the race 4 isolate 89-1A on Upland cotton and had similar symptom severity on Pima cotton. Karyotype profiling indicted an abundance of small chromosomes characteristic of karyotypes that include accessory chromosomes, with considerable variability between isolates. Collectively, our study indicates that Fov isolates from Alabama are genetically diverse, which may have been promoted by its persistence in cotton fields. [ABSTRACT FROM AUTHOR]