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The South China tigers (Panthera tigris amoyensis) are extinct in the wild, but viable populations remain in breeding centers and zoos after 60 years of effective conservation efforts. At present, however, the existing genetic variation of these tigers remains unknown. In this study, we assembled a high-quality chromosome-level genome using long-read sequences and re-sequenced 29 high-depth genomes of the South China tigers. We identified two significantly differentiated genomic ancestries in the extant populations, which also harbored some rare genetic variants introgressed from other subspecies, suggesting limited but essential genetic diversity to sustain the South China tigers. The unique pattern of dual ancestry and the genomic resources generated in our study pay the way for a genomics-informed conservation, following the real-time monitoring and controlled exchange of all reproductive South China tigers.