Fusarium oxysporum f. sp. vasinfectum (FOV) is a significant cotton (Gossypium spp.) pathogen causing vascular wilt, browning of the vascular tissues, and plant death in the most severe cases. This global disease is responsible for sizeable crop losses annually and is found in many cotton producing regions, including the Republic of Uzbekistan and the USA. Specifically, FOV race 4 (FOV4) has been disrupting production for years. This study aimed to genetically characterize FOV4 isolates causing disease in the main cotton producing region of Uzbekistan and compare with FOV4 isolates from the USA. A field study conducted in the Bukhara region of the Republic of Uzbekistan in the spring of 2022 identified both FOV4 and new Fusarium isolates from Upland cotton exhibiting typical Fusarium wilt symptoms. Molecular markers were initially used to identify isolates of interest, and a phylogenetic analysis was performed using partial EF1-α sequences, followed by a comparative genomic analysis. We also report for the first time the isolation of F. solani and F. commune causing Fusarium wilt in Uzbekistan. Furthermore, we show that the FOV4 population within our sampling region of Uzbekistan may be dominated by a single biotype with an effector profile similar to that of FOV race 7 (FOV7). One of these effector proteins is also present in the F. commune isolate showing virulence to cotton. Whole genome comparisons between FOV races can identify unique genetic markers for FOV4 and aid in the development of tools for breeding FOV resistant cotton varieties.