The Okinawa spiny rat, Tokudaia muenninki, is the only species with a Y chromosome in the genus Tokudaia. Its phylogenic relationship with two XO/XO species, Tokudaia osimensis and Tokudaia tokunoshimensis, lacking a Y chromosome and the mammalian sex-determining gene SRY, is unknown. Furthermore, there has been little cytogenetic analysis of the sex chromosomes in T. muenninki. Therefore, we constructed molecular phylogenetic trees with nucleotide sequences of cyt b, RAG1, and IRBP. All trees strongly supported that T. muenninki was the first to diverge from the Tokudaia ancestor, indicating that loss of the Y chromosome and SRY occurred in the common ancestor of the two XO/XO species after T. muenninki diverged. We found that the X and Y chromosomes of T. muenninki consisted of large euchromatic and heterochromatic regions by conducting G- and C-banding analyses. PCR, Southern blotting, and FISH revealed that T. muenninki males had multiple SRY copies on the long arm of the Y chromosome. At least three of 24 SRY sequences contained a complete open reading frame (ORF). A species-specific substitution from alanine to serine was found in all copies at the DNA-binding surface within the HMG-box, suggesting that it occurred in an original SRY.