Simple SummaryThe sex-determination pattern of reptiles is the most diverse among the amniotes. At present, there is still controversy about the sex-determination mechanism ofEremias multiocellata. We carried out genotyping by sequencing and bioinformatics analysis onE. multiocellata. The results show that it has genetic sex determination (GSD) under female heterogamety, which also shows that the sex chromosome is generally conservative in the lacertid lizards.AbstractThe multi-ocellated racerunner,Eremias multiocellata, 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 theE. multiocellata, 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 ofE. multiocellata, which support the GSD inE. multiocellatawithout geographic variation. To test the conservation of sex chromosome inEremias, the eight markers were further cross-tested by PCR amplification in 10 individuals of the Mongolian racerunner (Eremias argus), two of which exhibited cross-utility. The novel sex-linked markers could be mapped on the W chromosome of the sand lizard (Lacerta agilis). 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.