Stripe rust, caused by the fungal pathogen Puccina striiformis f. sp. tritici (Pst) is an important wheat disease in Canada since 2000. The virulence of at least 80 different isolates of this pathogen was evaluated on 18 near-isogenic wheat lines in the Avocet background. These isolates were mainly collected from western Canada and are representing the pathogen populations before and after the year 2000. Wheat seedlings were inoculated with the pathogen using a spore/talc mixture (ratio 1:20). The second leaf of infected plants were rated for infection types (ITs), 18–21 days after inoculation based on a scale of 0–9. At least, 39 different virulence patterns were identified. None of the races were virulent on Yr1, Yr5, Yr15. The gene Yr76 remain effective against all tested isolates since 1984, and YrSp was defeated infrequently by new and old isolates. While Yr6, Yr8 and Yr9 genes were effective against most Pst isolates between 1984-1994, but were defeated by most recent isolates. The results confirm the major shift in stripe rust populations toward a wider virulence spectrum in Canada since 2000.