A comprehensive physical map was generated for Ovis aries chromosome X (OARX) based on a cytogenomics approach. DNA probes were prepared from bacterial artificial chromosome (BAC) clones from the CHORI-243 sheep library and were assigned to G-banded metaphase spreads via fluorescence in-situ hybridization (FISH). A total of 22 BACs gave a single hybridization signal to the X chromosome and were assigned out of 32 tested. The positioned BACs contained 16 genes and a microsatellite marker which represent new cytogenetically mapped loci in the sheep genome. The gene and microsatellite loci serve to anchor between the existing radiation hybrid (RH) and virtual sheep genome (VSG) maps to the cytogenetic OARX map, whilst the BACs themselves also serve as anchors between the VSG and the cytogenetic maps. An additional 17 links between the RH and cytogenetic maps are provided by BAC end sequence (BES) derived markers that have also been positioned on the RH map. Comparison of the map orders for the cytogenetic, RH, and virtual maps reveals that the orders for the cytogenetic and RH maps are most similar, with only one locus, represented by BAC CH243-330E18, mapping to relatively different positions. Several discrepancies, including an inverted segment are found when comparing both the cytogenetic and RH maps with the virtual map. These discrepancies highlight the value of using physical mapping methods to inform the process of future in silico map construction. A detailed comparative analysis of sheep, human, and cattle mapping data allowed the construction of a comparative map that confirms and expands the knowledge about evolutionary conservation and break points between the X chromosomes of the three mammalian species.