Abstract: The study was conducted for the virulence analysis of the stripe rust disease samples collected from different wheat growing areas of Pakistan. Samples were selected out of CDRP sample collection 2004-05. The inoculum was increased and maintained on universally susceptible cultivar Morocco, inoculated at two leaf stage with urediospores under control conditions. Inoculum was collected separately for each disease sample in No. 0 capsule using a separate cyclone collector for each sample attached to a vacuum pump. Uredioospores were collected and inoculated on seedlings of differential genotypes sets. Samples analyzed for virulence showed the presence of virulence even against the breeding material considered to have resistant genes. The study also revealed the high disease response of commercial varieties against stripe rust. Virulence analysis can provide an early information to the breeders about susceptibility of their material and they can incorporate resistance gene to avoid future epidemic.