We applied a noninvasive prenatal test for the determination of fetal gender in multiple pregnancies by using free fetal DNA circulating in maternal blood in order to evaluate whether the quantification of male DNA could distinguish the fetal gender and the number of male and female fetuses in multiple pregnancies. We enrolled consecutively 44 women with twin pregnancies between 11–14 weeks of gestation. Peripheral maternal blood was collected, and genomic DNA was extracted from maternal plasma and analyzed for the multicopy DYS 14 sequence by using real-time PCR to quantify male DNA. Results showed that male DNA concentration was significantly higher in twin pregnancies with at least one male fetus, compared to twin pregnancies with only female fetuses. Comparing male DNA concentration in pregnancies with two male fetuses versus pregnancies with one female fetus and one male fetus, we did not obtain a significant difference between the two groups due to a slight overlapping of the range values. Therefore, our test correctly predicted fetal gender, distinguishing twin pregnancies with at least one male fetus from twin pregnancies with only female fetuses, with a diagnostic accuracy of 100%. For distinguishing pregnancies with two male fetuses from pregnancies with both female and male fetuses, a diagnostic accuracy of 76.1% was achieved.