True hermaphroditism is a disorder of sexual differentiation characterized by the presence of ovarian and testicular tissue in a person and it determines an increased risk for developing gonad cancer. Diagnostic and medical-surgical procedures with opportunity and successful in true hermaphroditism, facilitate the prevention of gonadoblastoma, besides contributing to solving social problem for assignation of sex. In this paper we present two cases with true hermaphroditism that had cytogenetic analysis and search for sequences of chromosome Y. Both patients, had a karyotype 46,XX and showed positive polymorphic regions in chromosome Y. The molecular detection of the Y chromosome is a non-invasive and fast procedure that allows explore cell lines by cytogenetic analysis. We conclude that this procedure is useful as it streamlines the management of disorders of sexual differentiation to identify individuals with higher risk to gonad cancer.