We have prospectively examined the diagnostic accuracy of a conventional multiplex polymerase chain reaction (PCR) analysis of maternal plasma for fetal gender determination in daily practice. Plasma DNAs were obtained from 168 pregnant women between five and 32 weeks of gestation. The 198-bp-specific sequence on Y-chromosome and the 261 bp ATL1-gene-specific sequence on X-chromosome were coamplified in a multiplex nested PCR manner. The results of fetal sex prediction were compared with routine analysis of fetal tissues and birth outcomes. The ATL1-specific sequences were detected in all cases but Y-chromosome-specific signals were observed in 95 of 97 plasma samples of women carrying male fetuses, leading to the sensitivity of 97.9, 100 and 100% for the first, second and third trimesters, respectively, and the specificity of 100% at all gestational stages. Therefore, fetal gender detection in maternal plasma with a conventional multiplex PCR assay is highly accurate in daily practice which should prove useful for non-invasive prenatal screening of sex-linked genetic disorders.