Puccinia striiformis f. sp. tritici (Pst), the stripe rust pathogen infecting cereal crops and grasses, was believed to have a hemicyclic life cycle consisting of uredinial and telial stages before the recent discovery of barberry (Berberis spp.) as an alternate (aecial) host for the fungus. This discovery has improved the understanding of the biology of the stripe rust pathogen. The Himalayan and near-Himalayan regions of Pakistan, China, and Nepal are considered as the center of diversity for Pst pathogen. High genetic diversity has been reported in these areas, probably resulting from the sexual reproduction of the stripe rust fungus. To determine if Berberis species growing in Pakistan are susceptible to Pst, we collected seeds of five species and two subspecies from the Himalayan region in 2016 and inoculated the seedlings with germinated teliospores of a Pakistani Pst isolate under controlled conditions. Pycnia and aecia were produced on all inoculated plants of these species and subspecies, and were demonstrated as Pst by successful infection of wheat plants with aeciospores. This study showed that the tested Pakistani Berberis species and subspecies are susceptible to Pst under controlled conditions.