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To determine the loss rate after amniocentesis in twins.This cohort study evaluated twin pregnancies with serum screening through the California Prenatal Screening Program. The primary outcome was loss of one or both twins at any gestational age. Exclusions were chromosomal/structural abnormalities, selective fetal reduction, terminations, neonatal deaths, ovum donation, and incomplete data. Loss rates were compared between three groups: (a) screen negative and no amniocentesis, (b) screen positive and accepted, or (c) declined amniocentesis. Multivariate logistic analysis generated adjusted odds ratios (aOR).Thirty-six thousand eight hundred twenty-one twin pregnancies had screening: 2698 (7.3%) were screen positive for aneuploidy or neural tube defects (NTD). Among screen-positive women, 861 (31.9%) were offered amniocentesis and 274 (31.8%) accepted. The post-procedure loss rate among screen-negative women was lower than among screen-positive women (3.0% vs 7.4%; P < .001; aOR 2.62; 95% CI, 1.16-2.99). Among screen-positive women, the loss rate was similar for those who underwent amniocentesis and for those who declined (8.8% vs 6.8%; .32; aOR 1.32; 95% CI, 0.66-1.91).Twins that are screen positive for aneuploidy or NTD have an increased risk of pregnancy loss. Those who are screen positive and undergo amniocentesis do not have an increased loss rate.