Human identification means the determination of a person's individuality. Various methods are employed for sex determination analysis in forensic investigations including visual, clinical, microscopic, and advanced techniques. DNA is the molecule of choice for forensic analysis. Forensic odontology is a fast-growing and promising branch of forensic medicine, it is a vital and integral part of forensic science that is most widely utilized for solving many crimes. The current study aims to assess the reliability of using the teeth pulp tissue samples for sex determination regardless of the type of teeth (milk or permanent), using Multiplex PCR targeting the SRY protein and AR genes in male and female samples. Results: The PCR analysis on blood samples confirmed that these blood samples belonged to 42 males and 33 females. SRY gene and SRY protein on the Y chromosome was shown at 778bp and 254bp respectively in 42 dental pulp tissue samples, while the androgen receptor gene (AR) on the X chromosome (AR) was at 293bp in all cases. One hundred percent of males showed negative Barr body with no false negative result, while 12.2% of females didn't show Barr body in dental pulp tissue samples and 87.8% showed positive Barr body in dental pulp tissue samples. sensitivity and specificity of the Barr body test on dental pulp tissue samples stained by H&E stain were 87.8% and 100% respectively. Conclusion: Dental pulp tissue is a significant source of sex determination.