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Ancestral polymorphisms are defined as variants that arose by mutation prior to the speciation event that generated the species in which they segregate. Their presence may complicate the interpretation of molecular data and lead to incorrect phylogenetic inferences. They may also be used to identify regions of the genome that are under balancing selection. It is thus important to take into account the contribution of ancestral polymorphisms to variability within species and divergence between species. Here, we extend and improve a method for estimation of the proportion of ancestral polymorphisms within a species, and apply it to a dataset of 33 X-linked and 34 autosomal protein-coding genes for which sequence polymorphism data are available in both Drosophila pseudoobscura and Drosophila miranda, using Drosophila affinis as an outgroup. We show that a substantial proportion of both X-linked and autosomal synonymous variants in these two species are ancestral, and that a small number of additional genes with unusually high sequence diversity seem to have an excess of ancestral polymorphisms, suggestive of balancing selection.