<p>Hybridization-based target enrichment sequencing (Hyb-Seq) is a powerful approach for phylogenomic studies of non-model organisms, particularly those with complex genomes or from degraded materials such as herbarium specimens. To improve phylogenetic resolution within the challenging Urticaceae family, we developed a new probe set, Urticaceae542, by combining the universal Angiosperms353 loci with lineage-specific loci from three representative Urticaceae genera. After evaluating its performance across 61 Urticaceae species, including herbarium specimens, we found that Urticaceae542 effectively facilitated both deep and shallow-scale phylogenomic analyses. The lineage-specific loci (Urti-MarkerMiner) provided more parsimony-informative sites and longer sequences, providing greater resolution for recent divergence events. In contrast, the Angiosperms353-derived loci (Urti-Angiosperms353) offered higher locus recovery efficiency and reduced missing data, features crucial for robust deep-level phylogenetic relationships. Moreover, the performance of Urticaceae542 was not significantly affected by sample age or preservation condition, but its efficiency varied significantly across different taxonomic clades and genera. Our findings demonstrate that combining universal and taxon-specific probes generates a robust strategy for resolving complex evolutionary histories in challenging plant families, and the Urticaceae542 provides a powerful new resource for phylogenomics of Urticaceae.</p>