Hybrid oospores are formed as a result of the cross of the A1 and A2 two mating types of oomycetes Ph. infestans. The aim of this study was the density defference of hybrid oospores in the detached leaves of the various layers of potato plant in wet chambers. In spring 2017, during the several visits on the potato fields of the western regions of Golestan province, samples with blight symptoms were collected and transferred to the laboratory. Isolation and purification of pathogen were conducted on rye B agar culture media with tetracycline antibiotics and benomyl fungicide at 500 ppm. Ph. infestans isolates were paired with standard tester strains A1 Ph. infestans on rye B agar with tomato juice. The mating type of each isolate by observing hybrid oospore was determined A2 and conversely, A1. Three pairs of A1 and A2 isolates with oospore production index at very high (OPI=4), on the base of examination of 10 light microscopic fields (0.2 mm2) at 10x objective lense were selected. Zoosporangium suspension was prepared from the edge of 7-day-old culture of each isolate in sterile distilled water and placed in a refrigerator at 4°C for two hours to leave the zoosporangiospores. Inoculum of each isolate was prepared in concentration of 5-7 zoosporangia. Before the flowering stage, in total 45 healthy leaflets were selected from the lower layers of potato plants of sensitive cultivar Agria, 45 leaflets of the middle and upper layers and after surface sterilization with 70% ethanol and 5% sodium hypochlorite, for infestation, were placed upside down in wet chambers (Petri dishes). Each leaflet was inoculated with one drop (20 µL) of zoosporangiospore suspension from both A1 and A2 mating types using a Pasteur pipet. The experiment was carried out in a completely randomized design with nine suspensions (treatment) and five replications. Inoculated Petri dishes were incubated at 18°C for 20 days in the dark conditions. The small pieces of each leaflet (5-10 mm2) were prepared from the lesion and it’s margin. To observe clearly oospores in the mesophyll, the samples were dyed using 96% ethanol and 10% sodium hypochlorite. The results of the study showed a significant difference of OPI at (p <0.001) between the lower and middle layers leaves with the upper of each plant. The highest density of oospore (OPI=4) was observed in the lower and middle layers leaves and lowest (OPI=1) in the upper layers leaves of each plant. There was on significant difference in OPI between the lower and middle layers leaves at (p <0.05). The results of this study will be useful for adjusting the levels of fungicide distribution between the upper, middle and lower layers leaves of potato plants in the fields.