Root rot is a serious disease of garlic in the Shandong Province of China. Infected plants display symptoms such as dwarfed garlic bulbs, yellowing of leaves starting from the periole, discoloration of the basal stem, and rot. The aim of this study was the identification of the main pathogens involved in garlic root rot. Fifty garlic plants with root rot were collected from the five main production areas of garlic in the Shandong Province. Isolates was incubated using selective medium and identified using morphological method and the ribosomal DNA internal transcribed spacer (ITS) sequence alignment, followed by the confirmation of pathogenicity and the identification of biological characteristics of the putative pathogens. The isolation frequency of Pythium isolated from garlic root was 45%, resulting in a total of 12 isolates. According to morphological identification and ITS sequence alignment, the 12 isolates were further identified as Pythium sylvaticum Compbell & Hendrix, Pythium heterothallicum Compbell & Hendrix, Pythium paroecandrum Drechsler, and Pythium violae Chesters & Hickman. All 12 isolates were pathogenic when inoculated on the root collar and roots of garlic plants. The optimal temperature of Pythium growth was 20–25 °C and pH was 6–7. Pythium sylvaticum, Pythium heterothallicum, Pythium paroecandrum, Pythium violae, and Pythium sp. 845 us-2 were identified as causal agents of garlic root rot.