Inherited disorders are one of the reasons of infertility and economic losses for the equine industry. The detection rate of chromosomal abnormalities is rising due to the use of sex chromosome linked molecular markers. Here, a rare sterile five years-old Arabian mare with XY male chromosomes is presented. The phenotype was corresponded to female with normal external genitalia. By transrectal ultrasound, there were hypoplastic ovaries and aplasia of uterine horns. Chromosome analysis was accomplished on blood samples using conventional, and G-banding techniques and confirmed by PCR and hormonal analysis. Although the karyotype (2n = 64, XY) revealed a typical male arrangement, it was SRY gene negative and amelogenin gene positive for X and Y chromosome. Hormonal analysis showed slight estrogenic activity of the ovary, but low progesterone and anti-mullerian hormones levels. In conclusion, this case of sex reversal mare (SRY-negative 64, XY) was recorded using cytogenetic, genetic, and hormonal analysis. Cytogenetics and molecular screening could be used as a fast approach for reproductive disorders evaluation in equine to save money, effort, and time of breeders.