Abstract: 【Objectives】 Seedling root morphology is closely related to crop nutrient uptakes and lodging resistance. To achieve maximum yield and nitrogen-use efficiency, the response of root growth and nitrogen uptake to N supply needs to be assessed for different oat cultivars.【Methods】 Three oat cultivars, namely Bayou 3, Bayou 9 and 200215, were selected as the test materials. The oat seeds were germinated and grew to the true leaf unfolded. Ten seedlings with the same size were picked up from each cultivar and transplanted into Hoagland’s nutrition solution, in which, low, middle and high NO-3-N concentration of 1, 3 and 6 mmol/L were designed. After three weeks’ culture, the seedlings with uniform growth were sampled from each treatment, the N contents in toots were measured with NMT technology, the root morphology was measured with root image analysis system WinRhizo. 【Results】 The study has shown that the NO-3-N concentrations have no significant impact on number of nodal roots for all the oat varieties, whereas they have significant impact on the lateral root density, total root length, root mean diameter and root distribution. Changes in NO-3-N concentrations have a significant impact on nitrogen absorption of Bayou No.3; however insignificant on Bayou No.9 and 200215. There is a consistency between dynamic change in NO-3 uptake flux and change in the number of nodal roots. NO-3 uptake flux is consistent with the total root length, root mean diameter ≤0.16mm and percentage of fine roots. 【Conclusions】 NO-3-N supply mainly affects oat lateral root (diameter ≤0.16mm) formation and growth in the seedling stage; it affects nitrogen uptake flux of three oat varieties. Changes in NO-3 uptake flux of roots were mainly affected by the total root length, root mean diameter and percentage of fine roots.