The majority of heifers born co-twins are infertile freemartins. Freemartins are sterile heifers with mostly XX chromosome constitution whose external genitalia appear to be masculinized. The present study was undertaken to investigate cytogenetic characteristics of bovine freemartins and their co-twins. Chromosome analysis was performed using peripheral blood lymphocytes obtained from a total of 18 Holstein and Hanwoo heterosexual co-twins. From the lymphocytic karyotype analysis, all but a pair of twins showed sex chromosome chimerism, XX/XY. However, the XX/XY ratio among individuals was widely variable and the XX/XY ratio was not changed during growth and development of a pair of twins from which multiple blood samples were analyzed. In conclusion our data showed that all freemartins and their co-twins were identified to have the XX/XY chimerism of sex chromosomes. The XX/XY chimerism is believed to have resulted from cell migration from the counter-sex by virtue of placentary anastomosis at an undefined stage of fetal development.