A SRY-negative XX male describes a female karyotype with testicular tissue resulting from abnormal gonadal development. In such cases male gonad formation takes place in the absence of a Y chromosome which supports the hypothesis that some other genes are also responsible for testicular development. The present case report describes a one-year-old dog brought to the hospital with a complaint of no oestrus cycle. Apparently normal masculine bitch had an enlarged clitoris with an os on radiograph. A gonado-hysterectomy was performed. Instead of ovarian structures both testicular masses were present and a normal complete bicornuate uterus with a normal cervix was observed. The histology of both gonads showed two testes, each with its ductus deferens and the uterus with typical histological structure. Cytogenetic evaluation of peripheral blood lymphocytes revealed a female with a normal karyotype (78, XX). Polymerase chain reaction (PCR) analysis of genomic DNA showed that the SRY gene was absent.