Autotoxicity restricts reseeding of new alfalfa(Medicago sativa L.) after alfalfa until autotoxic chemical(s) breaks down or is dispersed into external environments, often requiring up to a year or more. One solution for reducing autotoxicity would be to select germplasms or cultivars with tolerance to the autotoxic chemical(5) and use genetically breeding program. Bioassay of seed germination and early seedling growth was conducted to evaluate autotoxic responses of 3 varieties of alfalfa to the water-soluble extracts(at 4 and 8g/L) from alfalfa ‘Cody’leaf by using agar and filter paper medium in a petri-dish assay. Root length at 5 days after seeding was more sensitive to the extract than was hypocotyl length or seed germination, and was a better parameter of autotoxic effects of alfalfa leaf extracts. Use of an agar medium gave better sensitivity of root length than did use of filter paper. Evaluating tolerance with percent of control was more important indicator than was mean of root length because of significant variation among varieties in root length of control treatment. Bioassay ranked varieties in the following order of tolerance on the basis of relative root length; “Cody” >“ Pioneer 5373” >“ Alfagraze”. Seedling growth from old “Cody” seed was more sensitive to the autotoxic chemical(5) than was that from newly produced seed.