Fusarium graminearum causes head blight (FHB)and crown rot (CR)diseases in wheat. Compared with FHB, CR symptom development occurs slowly, usually taking 4 to 8 weeks to become visible. To characterize CR development, we used histological and real-time quantitative polymerase chain reaction analyses to assess fungal colonization during a timecourse of infection. Three distinct phases of infection were identified: i)initial spore germination with formation of a superficial hyphal mat at the inoculation point, ii)colonization of the adaxial epidermis of the outer leaf sheath and mycelial growth from the inoculation point to the crown, concomitant with a drop in fungal biomass, and iii)extensive colonization of the internal crown tissue. Fungal gene expression was examined during each phase using Affymetrix GeneChips. In total, 1,839 F. graminearum genes were significantly upregulated, including some known FHB virulence genes (e.g., TRI5 and TRI14), and 2,649 genes were significantly downregulated in planta compared with axenically cultured mycelia. Global comparisons of fungal gene expression with published data for FHB showed significant similarities between early stages of FHB and CR. These results indicate that CR disease development involves distinct phases of colonization, each of which is associated with a different fungal gene expression program.