Fusarium crown rot (FCR), caused by Fusarium culmorum (Wm.G.Sm) Sacc., is an important disease of wheat both in Iraq and other regions of wheat production worldwide. Changes in environmental conditions and cultural practices such as crop rotation generate stress on pathogen populations leading to the evolution of new strains that can tolerate more stressful environments. This study aimed to investigate the genetic diversity among isolates of F. culmorum in Iraq. Twenty-nine samples were collected from different regions of wheat cultivation in Iraq to investigate the pathogenicity and genetic diversity of F. culmorum using the repetitive extragenic palindromic (REP-PCR) technique. Among the 29 isolates of F. culmorum examined for pathogenicity, 96% were pathogenic to wheat at the seedling stage. The most aggressive isolate, from Baghdad, was IF 0021 at 0.890 on the FCR severity index. Three primer sets were used to assess the genotypic diversity via REP, ERIC and BOX elements. The amplicon sizes ranged from 200–800 bp for BOX-ERIC2, 110–1100 bp for ERIC-ERIC2 and 200–1300 bp for REP. A total of 410 markers were polymorphic, including 106 for BOX, 175 for ERIC and 129 for the REP. Genetic similarity was calculated by comparing markers according to minimum variance (Squared Euclidean). Clustering analysis generated two major groups, group 1 with two subgroups 1a and 1b with 5 and 12 isolates, respectively, and group 2 with two subgroups 2a and 2b with 3 and 9 isolates, respectively. This is the first study in this field that has been reported in Iraq.