A high-throughput and reliable seedling bioassay to screen wheat germplasm for crown rot resistance was developed. Single wheat seedlings were grown in square seedling punnets in a glasshouse and inoculated with a monoconidial Fusarium pseudograminearum isolate 10 days after emergence. The punnets were laid horizontally on their side and a 10-mu L inoculum droplet placed on the stem base. Seedlings were incubated at near-saturated relative humidity, and crown rot severity was assessed 35 days after inoculation. Studies on the duration of incubation period, inoculum concentration and temperature were carried out to optimize these parameters. Seedling growth at 25/15(+/- 5)degrees C in a glasshouse and 48-h incubation at near-saturated RH in darkness gave the best results. When crown rot resistance rankings of 16 Australian cultivars from the bioassay were compared with their field performance, Spearman's rank correlation coefficient was highly significant. This indicated that the seedling bioassay mimicked field resistance to crown rot in adult plants. A bootstrap resampling analysis showed little or no improvement in the coefficient of variation with an increasing number of replications, indicating a high level of precision and reproducibility. By detecting small but consistent differences in crown rot severity, the bioassay proved effective in large-scale screening for partial resistance: already over 1400 wheat genotypes have been screened. The high degree of precision makes this an invaluable tool in the understanding of pathogen aggressiveness, host specialization and parasitic fitness.