Understanding the genetic structure of a population is essential for the effective conservation of threatened species. This study presents the first analysis of the population genetic structure of the spider-tailed viper (Pseudocerastes urarachnoides) in western Iran, based on Cytb and CR1 mitochondrial gene sequences. Using spatial analysis of molecular variance and principal coordinates analysis, we delineated five distinct populations. We observed a pattern of low nucleotide diversity (π) and high haplotype diversity (Hd), with significant genetic differentiation between populations (ΦST = 0.1–0.92, P < 0.05). Intra-population genetic diversity was significantly lower than inter-population diversity (P < 0.001). The haplotype network analysis confirmed the existence of five distinct haplogroups for Cytb, four for CR1, and the combined dataset. This reveals a general lack of genetic connectivity between these populations. Although neutrality tests (Tajima’s D, Fu’s Fs) and the raggedness index suggested population stability, a pattern of isolation by distance was evident across the species‘ range. These results define five key management units for P. urarachnoides. As a unique endemic species facing significant threats, these findings provide the Iranian Department of Environment with a critical genetic foundation on which to develop targeted conservation strategies to preserve this viper and its associated ecosystems.
