In 1949, Canadian anatomist Murray Barr announced the discovery of a peculiar entity in the cell nucleus that was present in females and absent in males. The identity of this entity remained uncertain for a decade even though Barr hypothesized a relationship between it and the sex chromosomes and called it the “sex chromatin.” This hypothesis inspired the development of the chromatin into a technology that could indicate “chromosomal” or “genetic” sex, which supposedly established male and female sex difference as a binary and fundamental characteristic of humans and other animals at conception. Barr collaborated with other researchers and potential patients who applied the sex chromatin test, hoping that it could identify the “true” sex of intersexuals, homosexuals, and transsexuals. Ironically, the application of the test to intersexuals would lead to a revision of the identity of the sex chromatin itself. The history of the sex chromatin illuminates how the significance and essence of this laboratory object evolved with its use as a clinical and research tool. Researchers had hoped that the test would sort the intersex into just two categories, male and female. Instead, the sex chromatin helped to multiply categories of the intersex, distinguished them from inverts, underpinned psychosocial gender as a new dimension of sex difference, and in the process had its own identity refashioned. Today, we call it the Barr body and its story reminds us of the power and limit of biotechnologies to determine who we are.