Background: The infraorder Gekkota is intriguing because it contains multiple chromosomal and environmental sex determination systems that vary even among closely related taxa. Here, we compare male and females karyotypes of the pink-tailed worm-lizard (Aprasia parapulchella), a small legless lizard belonging to the endemic Australian family Pygopodidae. Results: We applied comparative genomic hybridization to reveal an XX/XY sex chromosome system in which the Y chromosome is highly differentiated from the X in both gross morphology and DNA sequence. In addition, FISH mapping has revealed that two microsatellite repeat motifs, (AGAT)n and (AC)n, have been amplified multiple times on the Y chromosome. Conclusion: XY karyotypes are found in other pygopodids (Delma inornata and Lialis burtonis), suggesting that the common ancestor of Pygopodidae also had XY sex chromosomes. However, the morphology and size of the Y chromosomes are different among the three species, suggesting that the processes underlying the evolution of sex chromosomes in the Pygopodidae involved chromosome rearrangements and accumulation and amplification of repeats.