The Himalayan and near Himalayan region of Pakistan, China and Nepal was recently identified as the center of diversity of Pst. The Pakistani Himalayan populations were shown to be recombinant and possibly maintained through sexual reproduction on the alternate host, Berberis spp. To examine the role of Berberis spp. in supporting Puccinia spp. in the Himalayan region of Pakistan, 274 pycnial/aecial-infected Berberis leaves and 16 grass samples with uredinial infections were collected in the region from 2012 to 2014. Amplification of infected grass and Berberis spp. samples with EF, ITS region, and β-tubulin primers and subsequent species identification based on comparisons of the sequences to sequences in GenBank identified at least five Puccinia spp. viz., P. brachypodii, putative P. coronata-loli and P. coronati-agrostis, P. striiformis f. sp. dactylis (P. striiformoides), and P. striiformis on Berberis and grasses. This infers a role of Berberis as alternate hosts to Puccinia spp. in the Himalayan region of Pakistan, and in contributing to the overall diversity of these species in the region. Microsatellite characterization of Pst samples collected on wheat in 2013 and 2014 indicated an overall high diversity and recombinant population structure in the region. However, the low frequency of wheat-infecting P. striiformis isolates obtained from Berberis spp. necessitates ongoing investigation.