Sex development anomalies represent a group of congenital pathologies in which chromosomal, gonadal and anatomical sex differentiation is atypical. The aim of our study was to use molecular biology techniques to elucidate sex in cases of anomalies of sexual differentiation in Burkina Faso. This cross-sectional study took place from March 2023 to June 2023. Oral and blood samples were collected respectively using sterile swabs and stored on Swab Kits and NUCLEIcardTM (https://www.copangroup.com/product-ranges/nucleic-card/) were used to determine gonosome profiles. Extraction was carried out using the DNA Swap solution Kit and the DNA IQ System Kit, and a PowerPlex® 21 kit (Promega) for amplification. The Applied Biosystems 9700 thermal cycler was used for PCR followed by 36 cm capillary electrophoresis in the Applied Biosystems 3130 prism sequencer. Sequence files were analyzed using GeneMapper IDX v. 3.2 software. Seven (07) patients were registered during the study period. There were 4 cases of XX DSD or 46, XX DSD and 3 cases of XY DSD or 46, XY DSD. The median age of our patients was 16 years. Civilian sex was male in 4 cases and female in 3. The most frequent reason for consultation was micropenis in 3 cases, followed by primary amenorrhea and sex ambiguity.