The authors’ objective was to measure amniotic fluid amino acid concentrations in pregnant women diagnosed as having fetuses with skeletal dysplasia in the second trimester of pregnancy. Eighteen pregnant women who had fetuses with with skeletal dysplasia detected by ultrasonography (skeletal dysplasia) in the second trimester and 35 women who had abnormal triple screenings indicating an increased risk for Down syndrome, but had healthy fetuses (control group), were enrolled in the study. Amniotic fluid was obtained by amniocentesis. The chromosomal analysis of the study and control groups was normal. Levels of free amino acids and non-essential amino acids were measured in amniotic fluid samples using GC/FID free (physiological) amino acid kit by gas chromatography. The mean levels of essential amino acids (histidine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan, and valine) in amniotic fluid were found to be significantly lower in fetuses with skeletal dysplasia than in the control group (p < 0.05). The detection of significantly lower amino acid concentrations in the amniotic fluid of fetuses with a skeletal dysplasia compared to healthy fetuses suggests amino acid deficiency may play an etiological role in the pathogenesis of skeletal dysplasia.