[Background] In recent years, with the expansion of kiwifruit cultivation area, frequent occurrences of diseases have increasingly affected the yield and quality of kiwifruit. Phytophthora cactorum, P. cinnamomi and P. lateralis are a group of pathogens that cause the kiwifruit root rot disease. [Objective] The present study aimed to establish and optimize a multiplex quantitative real-time PCR assay for simultaneously detecting the three pathogenic Phytophthora species, and to investigate the distribution of these pathogens in the main production areas of kiwifruit. [Methods] The Ypt1 (ras-related protein gene) sequences were aligned to develop species-specific TaqMan probes and primers for P. cactorum, P. cinnamomi and P. lateralis, respectively. A multiplex quantitative real-time PCR assay was established and optimized, and the specificity and sensitivity were tested. Finally, the detection system was used to analyze the Ypt1 gene content of three pathogens from the rhizosphere soils in the main production areas of kiwifruit. [Results] HEX, FAM and ROX fluorescence signals were detected in the DNA samples of P. cactorum, P. cinnamomi and P. lateralis, respectively, but no fluorescence signals in those of their closely related and other soil-borne pathogens. The sensitivity was 100 fg for each pathogen. By assaying 166 rhizosphere soil samples of kiwifruit plants from Zhouzhi and Meixian Prefecture of Shaanxi Province, P. cactorum was found to be the most widely distributed along with the highest Ypt1 gene content, while P. cinnamomi and P. lateralis were relatively less frequent. [Conclusion] The established multiplex quantitative detection method was specific and sensitive, and was suitable for the detection and quantification of P. cactorum, P. cinnamomi and P. lateralis. This technique would be useful in early diagnosis, monitoring and prevention of Phytophthora disease in kiwifruits.