INTRODUCTION Serum biochemical marker concentrations in twin pregnancies reflect the presence of two fetuses rather than one. In general, the concentrations are approximately double those found in singleton pregnancies. The objective was to estimate the difference between levels of the two biochemical markers pregnancy-associated plasma protein-A (PAPP-A) and free β-human chorionic gonadotropin (free β-hCG) in twin pregnancies relative to singleton pregnancies and establish improved screening procedure for chromosomal anomalies such as trisomy 21 in twin pregnancies. METHODS A total of 4843 unaffected and 47 trisomy 21 affected twin pregnancies were included in the study. Chorionicity specific medians were generated for PAPP-A and free β-hCG from gestational age 8 to 14 weeks. Multiple of the median for each of the markers were calculated. Detection rates (DR) and false-positive rates (FPR) were estimated for screening tests incorporating Nuchal Translucency and maternal age, with and without biochemistry. RESULTS Medians for the two markers for monochorionic and dichorionic twins in unaffected pregnancies show a gestational age specific increase relative to the singleton medians. Allowing for gestation and chorionicity, twin pregnancies affected with trisomy 21 had higher levels of free β-hCG and lower levels of PAPP-A. Adding biochemistry into the risk assessment increased the DR for fetal trisomy 21 in dizygotic twin pregnancies from 78% to 90%, and decreased the FPR from 8.0% to 5.9%. DISCUSSION This study demonstrates that generation of chorionicity specific medians for the biochemical markers and their use in risk assessment can increase the performance of first trimester screening for chromosomal abnormalities in twins to a level comparable to that in singleton pregnancies. We believe that the results are sufficient to document the distribution of the two markers in twins and to include these data in routine risk assessment in twin pregnancies.