Invasive procedures including chorionic villus sampling and amniocentesis in sex-linked diseases increase the risk of fetal loss. Therefore, Noninvasive fetal gender determination using cell-free fetal DNA (cffDNA) in maternal plasma may be promising. Fifty pregnant females with gestational age ranging from six to ten weeks were included. cffDNA were extracted from maternal plasma and amplified by real time and conventional PCR for identification of SRY, DYS14 and DAZ genes as specific genetic markers for male-bearing pregnancies. In general, sensitivity and specificity of real time PCR was better than conventional PCR. However, sensitivity of DYS14 gene and specificity of SRY gene by real time PCR was equal to those of conventional PCR. Sensitivity of DYS14 gene was the highest and sensitivity of SRY gene was the lowest. However, combination of the three Y-chromosomal sequences in the diagnosis increased the accuracy of the test, which is suitable for clinical application.