Background/Objective: Several studies have shown that the 2nd-trimester maternal serum α-fetoprotein (AFP) level is significantly lower and that the maternal serum human chorionic gonadotropin (hCG) level is significantly higher in the presence of a female fetus. This may potentially affect Down syndrome (DS) screening such that a higher false-positive rate may occur in women carrying a female fetus, whereas a lower detection rate may result in those carrying males. The purpose of this study was to evaluate the gender impact on marker levels in DS pregnancies and its effect on DS screening. Methods: The study included 62 DS pregnancies with a single fetus of known gender (31 male and 31 female). Only pregnancies with chromosomal analysis showing trisomy 21 were included. The maternal serum levels of hCG, AFP, and unconjugated estriol were measured at 16–20 weeks of pregnancy. These levels were expressed as gestational-age-corrected multiples of the median. Results: No statistically significant differences were noted in maternal serum levels of hCG or AFP in DS pregnancies between women carrying a female and those carrying a male DS fetus. No statistically significant differences in ‘screen-negative’ rates were noted among male and female fetuses. Conclusions: In normal pregnancies, the maternal serum hCG level is higher, and the AFP level is lower in the presence of a female fetus. However, this gender-related difference is not apparent in DS pregnancies. Therefore, the gender-related differences in serum marker levels would not result in a lower detection rate of DS in male fetuses.