Background: The passage of nucleated cells between mother and fetus is well recognized (Lo et al., 1989, 1996). As well as, cell-free fetal DNA in maternal plasma or serum is at present widely investigated as a source of fetal genetic material (Stanghellini et al., 2006) [18]. There has been much recent interest in the use of DNA derived from plasma or serum (Boland, 1996). This DNA can be utilized for molecular diagnosis as well as prenatal sex discernment. Objective: To establish an easy, reliable, and completely safe method for fetal gender determination alternative to conventional exhausting current techniques applied in gynecologic hospitals and clinics, besides its further applications in forensic casework. Methods: EDTA-Blood samples were taken from 30 pregnant women all in the third trimester of pregnancy, then plasma was separated from each sample, from which DNA was isolated using a QIAamp DNA Mini Kit, with special modifications done in the extracting procedure to concentrate and obtain minute quantities of fetal DNA, together with maternal DNA, from maternal plasma. In addition, bloodstain samples were taken from the husbands of women who were pregnant with male fetuses from which DNA was isolated using a QIAamp DNA Micro Kit for comparison. DNA quantification was done using a Real-time PCR utilizing Quantifiler Duo Kit. PCR was done using an AmpFlSTR Y-Filer Kit, then amplified products were typed using a 3130 Genetic Analyzer. Results: Full and partial Y-STR profiles (6–17 STR loci) were obtained from all plasma samples taken from pregnant women with male fetuses, while negative Y-STR profiles (no single STR locus) were obtained from all plasma samples taken from pregnant women with female fetuses. Conclusion: It is recommended to use Y-STR profiling as an alternative technique for fetal gender determination during the third trimester of pregnancy, in addition to its significance in forensic casework.