| Summary: | The multi-ocellated racerunner, <i>Eremias multiocellata</i>, was considered to have temperature-dependent sex determination (TSD), as its sex ratio can be influenced at different temperatures. However, such an observation contrasts with recent findings that suggest TSD is less common than previously thought. Here, a genotyping-by-sequencing (GBS) approach was employed to identify sex-linked markers in the <i>E. multiocellata</i>, for which the mechanism choice of TSD or GSD is still controversial. We preliminarily identified 119 sex-linked markers based on sex-associated sex-specific sequences, 97% of which indicated female heterogamety. After eliminating the false positives, 38 sex-linked markers were recognized, all of which showed the ZW/ZZ system. Then, eight of the novel markers were verified by PCR amplification from 15 populations of <i>E. multiocellata</i>, which support the GSD in <i>E. multiocellata</i> without geographic variation. To test the conservation of sex chromosome in <i>Eremias</i>, the eight markers were further cross-tested by PCR amplification in 10 individuals of the Mongolian racerunner (<i>Eremias argus</i>), two of which exhibited cross-utility. The novel sex-linked markers could be mapped on the W chromosome of the sand lizard (<i>Lacerta agilis</i>). Our finding that the sex-linked markers are shared in closely related species, along with a conserved synteny of the W chromosome, further supports the homology and conservation of sex chromosomes in the lacertid lizards.
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