Abstract: This study aims at exploring the causes of poor growth and short and small plant in the seedling stage of winter wheat, and studies the clear pathogens of this disease by using the conventional tissue separation and purification method of the root system of wheat to obtain two similar Pythium isolates, i.e. SDCY-1 and SDJY-1. In addition, the morphological characteristics, identification of pathogenicity and CO I sequence analysis of the results of the two isolates show that the two isolates of the white colonies developed mycelium, and the culture medium surface showed a large number of aerial hyphae. After artificial inoculation, mycelium can cause poor growth, short root, fresh weight reduction, and hygrophanous to brown color changes in wheat. The newly separated strain was similar to that of the original inoculation strain. According to the morphological characteristics and phylogenetic tree, the two isolates were the same species of Pythium, namely Pythium ultimum. The results of this study can provide a scientific basis for Pythium root rot control in winter wheat and resistance breeding.