ABSTRACT Understanding the dietary ecology of endangered carnivores is critical for their conservation and management, as it elucidates their interaction with prey, other co-occurring carnivores, and humans. We used DNA metabarcoding to examine the seasonal and spatial variation in the diet of dholes (Cuon alpinus) in Tadoba Andhari Tiger Reserve, a key stronghold for the species in central India. We evaluated a simpler non-invasive fecal sample collection method and optimized a novel blocking oligonucleotide to suppress host DNA amplification. Our results revealed that pinches of scat in lysis buffer, coupled with N-spacer-tagged primers for amplifying diet DNA, significantly improved prey detection, sequencing efficiency, and reduced host contamination compared to traditional whole-scat-in-silica methods. Across 169 genetically confirmed dhole scats collected over two different seasons, we identified 15 vertebrate prey species. Dhole diet was dominated by wild ungulates, especially chital (Axis axis) and sambar (Rusa unicolor), with negligible consumption of domestic species. However, we observed strong seasonal and spatial variation; monsoon diet was diverse with greater variability, while winter diet was more consistent and dominated by large-bodied prey. Core zone exhibited wider dietary profiles than buffer areas that included mixed use landscapes. These findings suggest that wild prey availability, habitat quality, and seasonal foraging constraints shape dhole dietary patterns. Our study underscores the value of improved sampling and sequencing strategies for carnivore dietary studies and highlights the importance of sustaining wild prey populations to ensure the long-term persistence of dholes in multi-use landscapes.