Increased fetal lung heterogeneity has been associated with term fetal lungs in singleton gestations. Theobjective of this study was to determine if fetal lung heterogeneity index (HI) differs between twin and singleton fetuses in the late second and third trimesters. Prospective cohort study of women with singleton and twin gestations with medically-indicated ultrasound examinations at 24weeks of gestation onward. Grayscale transverse fetal lung images were obtained at thelevel of the four-chamber heart. A region of interest wasselected in each fetal lung image. Fetal lung HI was determined with MATLAB software using a dithering technique with ultrasound image pixels transformed into a binary map form from which a dynamic range value was determined. HI averages and standard deviations were generated for twin and singleton fetuses from 24weeks gestation onward. Two sample t-tests were used to compare the mean HI at each gestational week between singleton and twin fetuses. In total, 388 singleton and 478 twin images were analyzed. From 35 through 38weeks of gestation a statistically significant divergence in mean HI was observed withhigher means in singleton compared to twin fetuses. At24weeks of gestation there was a significantly higher HI in twin fetuses compared to singletons. No differences in fetal lung HI were observed between 25 and 34weeks gestational age. Differences in fetal lung HI were observed when comparing twin and singleton fetuses. Further investigation is required to determine the potential clinical significance of these findings.