Freemartinism syndrome is a case of sterility in female twin off spring when twin pregnancy exists with the different sexes, and caused by involvement of male twin's blood stem cell factor and anti-Mullerian hormone into female twin. Freemartinism may develop in female twin at a rate of 82.5% and 92% in twins with different sexes. Different methods are used to determine freemartinism syndrome. However, early identification of freemartinism is very important to determine the potential of calf for breeding purposes. Early identification of freemartinism could be accomplished by cytogenetic and molecular methods via Y-specific primers. However, molecular methods are preferred due to their accuracy, ease of application and shorter duration of process. In this study, freemartinism status of three female calves born with twin male calves was investigated by polymerase chain reaction (PCR). PCR process was carried out by using two primer sets, specific to Y-chromosome. Moreover, the length of the vagina and reproductive tract was examined by transrectal ultrasonography. PCR process revealed that the two female calves had the Y chromosome, whereas the other calf did not have the Y chromosome. Furthermore, length of vagina is longer (9 cm) in a calf not having Y chromosome than the other calves, and ultrasound examination indicated the presence of cervix in a calf without Y chromosome. According to current results, it was concluded that PCR technique could be used to investigate freemartinism cases in female twin calves with male twins and non-pregnant females following recurrent breeding.