<p><i>Taxus wallichiana</i> is an endangered Himalayan Yew having great ecological and economic significance in the Western Himalayas but lacks adequate knowledge of conservation importance. Present study aimed to assess and map the spatial genetic diversity using simple sequence repeat (SSR) markers and understanding genetic structure for conservation implications. In total, 583 individual trees from 21 populations of Uttarakhand Himalayas were investigated with 10 SSR loci, which are highly polymorphic with a mean PIC value 0.77. As exemplified by observed heterozygosity (Ho = 0.42) and expected heterozygosity (He = 0.72), present study reports a high genetic diversity in the native sampled populations. Positive value of mean inbreeding coefficient indicated severe inbreeding and most evaluated SSR loci exhibited a significant departure from Hardy–Weinberg Equilibrium. Nevertheless, the populations are assessed in their steady state showing insignificant genetic differentiation (F_ST = 0.07), less robust clustering, and high genetic admixing. This indicates that the populations are genetically well connectivity with adequate gene flow (Nm = 3.6). Furthermore, overlaying of the genetic parameters revealed that populations in the Kumaon region distinguishingly showed a high genetic diversity, and the region could be considered as major diversity hotspots of the <i>T. wallichiana</i> in the Western Himalayas. Specifically, the populations, namely TW21, TW19, TW18, TW15, and TW12 could be considered on top priority for conservation. To the best of our knowledge, this is the first comprehensive genetic study carried out in any Himalayan Yews, and the information generated herein is novel and of paramount importance in guiding conservation decisions.</p>