Céspedes' sex was subject to dispute as a potentially intersex individual, leading to a trial by the Spanish Inquisition on charges of sodomy, witchcraft, transvestism, and bigamy.

[5]: 30 [4] [6][7]: 68  Born into slavery, and branded on the cheeks as the offspring of a slave,[7]: 68 [8][9] Céspedes was freed as a child, and took the surname of a former owner's wife.

Undeterred after release, Céspedes resumed dressing as a man and found work as a soldier, taking a role in the suppression of the Rebellion of the Alpujarras under the command of John of Austria.

[2]: 59–60  In 1586, when Céspedes was forty and Caño was twenty-four, the couple were finally married; they lived together in Yepes[2]: 59–60  in the vicinity of Toledo, Spain for a year.

[4][8][c][7]: 75 In June 1587, acting on a neighbor's accusation, the couple were arrested, charged with "sodomy",[4][10] and imprisoned in the municipal jail in Ocaña, Spain.

Céspedes argued this natural (intersex/hermaphroditic) condition also made the witchcraft charge, of having the devil's aid in appearing as a man or woman, unfounded.

[d][2]: 62–64  To explain the lack of visible evidence of a penis, Céspedes said it had been injured and amputated shortly before imprisonment, following a riding injury.

[5]: 31 [8] Céspedes was also subjected to a public humiliation, an auto-da-fé, being paraded around Toledo's central square in a sanbenito mitre and robes.

[3]: 24 Various historical and medical studies of Céspedes's case have attempted to classify the Spaniard as intersex, as transsexual, or as a hypospadic male; other authors have viewed Céspedes as a lesbian woman (who may have adopted male clothes to acquire more social freedom), as transgender (perhaps a trans man whose claims of being a "hermaphrodite" were attempts to explain his gender dysphoria without a specific word for it),[15] or as non-binary, defying a binary model of gender and sex.

[10][7]: 75  Lisa Vollendorf says that while "even when medical doctors provided contradictory evidence, the Inquisition maintained that sex was an indisputable material fact" (displaying, she says, "an almost fetishistic interest in Céspedes's genitalia"), Céspedes described not only his physiology but also gave "behavioral and psychological explanations for his masculinity" he had lived for decades, and drew on his knowledge of medicine and history and cited Aristotle, Augustine, Cicero, and Pliny in arguing that his intersex body was not "unnatural or unprecedented".