The sex chromatin or Barr body is a condensation of chromatin present at the nucleus of cells in female individuals. Their observation is possible in different cell types and is used for the rapid diagnosis of biological sex. The observation of this condensation in cells of the pulp tissue can be useful in the forensic diagnosis of sex, primarily because of the protection it give dental hard tissues, but its diagnostic value has not been tested. The purpose of this study is to analyze the diagnostic value of the observation of sex chromatin in cells of the pulp tissue. We used 40 premolars and third molars-20 from men with a mean age of 20.6 years (SD 8.8) and 20 from women with a mean age of 20.4 years (SD 7.4). The pulp was obtained from teeth extracted and localized 50 cells per plate. The presence of a cell with visible Barr body was considered positive for women. With these results, we evaluated the diagnostic performance of the test. The performance of the test was 100%; of the 50 cells observed per plate, mean Barr bodies positive cells was 20.4 (SD 0.44) in female samples. There was no positive cell in preparations of male subjects. The diagnostic test observation of sex chromatin in dental pulp is a reliable test for sex determination. This study provides a gold standard for performance assessment in which the teeth have suffered extreme physical conditions, such as incineration and dumping.