Yellow rust, caused by Puccinia striiformis f.sp. tritici (PST), is an important disease that threatens wheat production in Pakistan. This study was designed to explore the virulence and simple sequence repeat (SSR) diversity of the Pakistani PST population and the ongoing selective pressures of widely grown wheat cultivars. Analyses of 49 isolates sampled from the North-West Frontier Province of Pakistan led to the identification of 12 distinct pathotypes. The virulence frequencies of v2 (virulent against Yr2), v6, v7, v9, vSU and v27 ranged from 63% to 100%. Virulences v3, v4, v17 and vSD were uncommon, whilst v5, v10, v15, v24, v32 and vSp were not detected. The pathotypes thus described were then classified into 27 distinct genotypes. Bayesian structure analysis clustered these genotypes into five groups (in addition to one hybrid isolate) which represent three distinct lineages of the SSR-based phylogenetic tree. Of the studied isolates, 80%, represented by three predominant pathotypes (P1–P3), belonged to the same characteristic Pakistani lineage, whilst the other isolates were close to either a Mediterranean lineage or a Northern European lineage. Genetic recombination was detected within P2 isolates. Resistance genes postulated in 40 Pakistani wheat cultivars indicated the high frequency of Yr2, Yr6, Yr7, Yr9, Yr27 and YrSU. Only 11 cultivars were found to be resistant to P1–P3. Migration and varietal diversity factors might contribute to maintaining the currently high genetic diversity in Pakistani PST, and have serious regional implications for wheat improvement programmes.