Technical Abstract: Screening for Fusarium head blight (FHB) involves inoculating barley (Hordeum vulgare L.) seed heads with Fusarium graminearum [teleomorph Gibberella zeae] followed by visual observation of disease progression and analysis for the mycotoxin deoxynivalenol (DON). Disease symptoms and DON are not uniformly expressed and DON production varies with environment. DON and FHB are not highly correlated with one another. The objective of this study was to develop an enzyme linked immunosorbant assay (ELISA) for quantification of FHB in barley. Mononclonal antibodies raised to Fusarium graminearum were tested for cross-reaction with other Fusarium spp. Antibodies from cell line IF8 cross-reacted with all Fusarium spp. but not other ascomycota tested. A laboratory method was developed using seed lots with no, low, and high levels of DON. Varying quantities of seed, extraction buffer, and agitation were tested and Fusarium quantified with ELISA. The best protocol for ELISA used 5 g seed, a 1:6 (w/v) ratio of seed:extraction buffer for 1 h. Five genotypes each for high, medium, and low ELISA values were selected from a field experiment using a doubled-haploid mapping population in 2003. The lines were grown in 2004 and scored for FHB, DON and ELISA. ELISA had lower error than FHB or DON and lines low, medium, and high ELISA in 2003 had low, medium, and high DON, FHB, and ELISA in 2004. ELISA and DON were correlated in both controlled studies (r=0.51) and samples selected from grain elevators in 1993-2003 (r=0.71). The correlation among ELISA and DON in the grain elevator samples indicates naturally occurring Fusarium spp. outside the B clade had no effect on the performance of the ELISA analysis.