Maternal biologic factors can affect the fetal fraction in cell-free DNA-based prenatal screening assays, thereby limiting the effectiveness. Higher rates of indeterminate results because of a low fetal fraction have been described in cases of maternal autoimmune disease in pregnancy. Existing studies are confounded by the concomitant maternal use of anticoagulants, which may independently influence the test characteristics. This study aimed to evaluate the differences in fetal fraction, indeterminate results, and total cell-free DNA concentration among women with an autoimmune disease in comparison with controls, using our in-house developed, noninvasive prenatal screening platform in the absence of maternal anticoagulation use. This was a retrospective, single institution cohort study of a previously validated, cell-free DNA-based, noninvasive prenatal screening assay using a low-pass whole-genome sequencing platform between 2017 and 2019. A diagnosis of an autoimmune disease included systemic lupus erythematosus, rheumatoid arthritis, multiple sclerosis, inflammatory bowel disease, and others. Immunomodulator therapies included biologics, corticosteroids, hydroxychloroquine, azathioprine, and intravenous immunoglobulin. Women who were using anticoagulants were excluded. We evaluated the association between autoimmune disease and fetal cell-free DNA fraction, indeterminate results, and total cell-free DNA concentration using univariate and multivariate analyses, stratified according to immunomodulator therapy and adjusted for body mass index, fetal sex, and gestational age at sample collection. A total of 1445 patients met inclusion criteria. Of those, 43 women had a confirmed autoimmune disease, with 25 of those not on immunomodulator therapy and 18 on immunomodulator therapy. The mean fetal fraction for women with an autoimmune disease was significantly lower than for controls (9.7% vs 11.9%; P=.004). The rate of indeterminate results was significantly higher among women with an autoimmune disease than among controls (16.3% vs 3.5%; P<.001). The total cell-free DNA concentration was not statistically different between the groups (94.8 pg/µL for women with an autoimmune disease vs 83.9 pg/µL for controls; P=.06). In a logistic regression, women with an autoimmune disease had significantly higher odds of receiving an indeterminate result than controls, (adjusted odds ratio, 5.3; 95% confidence interval, 2.0-14.2). Linear regression analysis showed a significant negative association between having an autoimmune disease and the fetal cell-free DNA fraction (aβ, -2.1; 95% confidence interval, -3.4 to -0.6). When stratifying by treatment status, the mean fetal fraction was 9.8%, 9.6%, and 11.9% for women with an autoimmune disease not on immunomodulator therapy, women with an autoimmune disease on immunomodulator therapy, and the controls, respectively (P=.02). The rate of indeterminate results increased in a stepwise fashion from 3.5% to 11.1% to 20.0% for controls, women with an autoimmune disease on immunomodulator therapy, and women with an autoimmune disease not on immunomodulator therapy, respectively (P<.001). Logistic regression analysis demonstrated higher odds of an indeterminate resultfor women with an autoimmune disease not on immunomodulator therapy than for controls, (adjusted odds ratio, 7.3; 95% confidence interval, 2.3-22.5). There was a negative association between women with an autoimmune disease not on immunomodulator therapy and the fetal fraction when compared with controls (aβ, -2.2; 95% confidence interval, -4.2 to -0.3). Women with an autoimmune disease have lower fetal cell-free DNA fractions and higher rates of indeterminate results than women without an autoimmune disease. There was no difference in total cell-free DNA concentration. Treatment of maternal autoimmune diseases with immunomodulator therapy may decrease the indeterminate result rate.