Accurate discrimination between closely related mahseer species remains a persistent taxonomic challenge, particularly for Tor tambra and Tor tambroides, which exhibit strong morphological similarity and limited resolution using mitochondrial DNA markers. In this study, we generated high-quality hybrid genome assemblies for male and female T. tambra and performed genome-wide comparative analyses to identify nuclear loci capable of distinguishing these species. Despite high overall genomic similarity and strong reciprocal mapping rates, a mapping-based unmapped-read discovery framework identified candidate nuclear regions exhibiting consistent asymmetric read support between taxa. These analyses indicate that strongly asymmetric candidate regions are highly localized and, under the applied detection framework, represent only a small fraction of the reference genome. Phylogenomic reconstruction based on conserved Benchmarking Universal Single-Copy Orthologs robustly resolved T. tambra and T. tambroides as distinct, well-supported nuclear lineages, while Mash-based genome-wide comparisons revealed consistent but shallow divergence (≈1.5%), supporting recent evolutionary separation. Genome-informed primer design and multi-individual validation confirmed one robust speciesspecific nuclear marker and additional loci containing fixed interspecific nucleotide differences. Furthermore, k-mer-based analysis detected a female-biased enrichment signal under stringent filtering conditions, although this observation requires validation across multiple individuals. Collectively, these findings provide robust genome-scale evidence supporting species-level differentiation between T. tambra and T. tambroides, while demonstrating that divergence is highly localized within an otherwise conserved genomic background. Together with previous mitochondrial evidence showing limited discriminatory power for this species pair, this study establishes a reproducible nuclear genome-based framework for species delimitation and diagnostic marker development in recently diverged teleost lineages.

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