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Summary Australia was one of the earliest regions outside Africa to be colonized by fully modern humans, with archaeological evidence for human presence by 47,000 years ago (47 kya) widely accepted [ 1, 2 ]. However, the extent of subsequent human entry before the European colonial age is less clear. The dingo reached Australia about 4 kya, indirectly implying human contact, which some have linked to changes in language and stone tool technology to suggest substantial cultural changes at the same time [ 3 ]. Genetic data of two kinds have been proposed to support gene flow from the Indian subcontinent to Australia at this time, as well: first, signs of South Asian admixture in Aboriginal Australian genomes have been reported on the basis of genome-wide SNP data [ 4 ]; and second, a Y chromosome lineage designated haplogroup C ∗ , present in both India and Australia, was estimated to have a most recent common ancestor around 5 kya and to have entered Australia from India [ 5 ]. Here, we sequence 13 Aboriginal Australian Y chromosomes to re-investigate their divergence times from Y chromosomes in other continents, including a comparison of Aboriginal Australian and South Asian haplogroup C chromosomes. We find divergence times dating back to ∼50 kya, thus excluding the Y chromosome as providing evidence for recent gene flow from India into Australia. [ABSTRACT FROM AUTHOR]