Phytophthora root rot (PRR) caused by Phytophthora sojae, is one of the most destructive soybean diseases. The deployment of resistant cultivars is an important disease management strategy. To this aim, the development of a fast and effective method to evaluate soybean resistance to P. sojae is strategic. In this study, a detached-petiole inoculation technique was developed and its reliability was verified in soybean cultivars and segregant populations for PRR resistance. The detached-petiole and hypocotyl inoculation methods were used to assess the resistance of soybean cultivars, the F₂ population of a Zhonghuang47 × Xiu94-11 cross, and the derived F_2:3 population. The reactions of 13 analysed cultivars to three P. sojae isolates were consistent between the two inoculation techniques. The reactions of the F₂ and F_2:3 populations to isolate PsMC1 were 95.20% similar between the two inoculation methods. The segregation of the resistance and susceptibility fit a 3 : 1 ratio. Our results suggest that the detached-petiole technique is a reliable method, and reveal that the PRR resistance in Xiu94-11 is controlled by a single dominant gene. The phenotypic ratios of the tested Jikedou2 × Qichadou1 F₂ population using the detached-petiole inoculation technique fit a 3 : 1 ratio (Resistance : Susceptibility). This demonstrated that Qichadou1 contains a single dominant gene conferring resistance to P. sojae. Our new detached-petiole inoculation technique is effective, reliable, non-destructive to the plant, and does not require an excessive amount of seeds. It may be suitable for the largescale screening of soybean resistance to multiple P. sojae isolates.