Potato late blight (Phytophthora infestans) is an important disease causing severe damage in potato crop. One hundred forty nine single lesion isolates of P. infestans collected from naturally different potato growing areas of Punjab province during 2017-18 were characterized for pathotype determination based on their lesion area and infected area percentage after their inoculation onto detached leaves of potato. The results showed that large variations in virulence were present among isolates for each regional P. infestans population studied. Those isolates collected during 2017-18 exhibited 69.44% and 70.12% virulence percentage respectively. The highest lesion length and infected percentage area of isolate Oka-24 recorded 44.33 mm (63.33%) while lowest was 1 mm (1.43%) shown by isolate Srg-5 during both years. This is the first comprehensive study to determine pathotype of isolates in Pakistan. The experimental findings indicated that population of P. infestans in the Punjab province comprises diverse isolates with low to high virulence potential as observed.