Citrus is one of the major economic horticultural crops of India. In recent years, the production of citrus is greatly affected because of diseases caused by Phytophthora, Citrus Tristeza Virus (CTV), etc. Among them, Phytophthora causes 80% to 90% economic losses to grower. It has an extensive host range infecting different crops and weeds. It is very difficult for control management of this pathogen because of its pseudofungus nature. For control management, there is a need of sensitive and accurate diagnosis of the disease. In this study, internal transcribed spacer (ITS) regions are used to discriminate Phytophthora from other fungi because ITS region sequences are much more variable, even among very closely related organisms. More than 25 restriction enzymes are used for the digestion of the PCR product. By using restriction digestion of the amplified PCR product of ITS4-ITS6, it was found that there is clear-cut discrimination where it is possible to identify the presence of Phytophthora from a bulk of fungi samples. Further, specific amplicon sequence analysis was made for designing specific primer for Phytophthora. This primer was used for the detection of Phytophthora in the bulk of fungi isolates. By applying this accurate diagnosis method, the grower can follow appropriate control management practices. Further, there will not be mismanagement in disease control. This method is applied to soil as well as nursery plant to detect infection of Phytophthora. It is easy for a pathologist to diagnosis disease and develop accurate control management practices.