Stress exposure during gestation is implicated in several neuropsychiatric conditions, including autism spectrum disorder (ASD). Previous research showed that prenatal stress increases risk for ASD with peak exposure during the end of the second and the beginning of the third trimester. However, exposures to prenatal stress do not always result in ASD, suggesting that other factors may interact with environmental stressors to increase ASD risk. The present study examined a maternal genetic variation in the promoter region of the serotonin transporter gene (5‐HTTLPR) affecting stress tolerance and its interaction with the effect of environmental stressors on risk for ASD. Two independent cohorts of mothers of ASD children recruited by the University of Missouri and Queen's University were surveyed regarding the prenatal environment and genotyping on 5‐HTTLPR was performed to explore this relationship. In both samples, mothers of children with ASD carrying the stress susceptible short allele variant of 5‐HTTLPR experienced a greater number of stressors and greater stress severity when compared to mothers carrying the long allele variant. The temporal peak of stressors during gestation in these mothers was consistent with previous findings. Additionally, increased exposure to prenatal stress was not reported in the pregnancies of typically developing siblings from the same mothers, regardless of maternal genotype, suggesting against the possibility that the short allele might increase the recall of stress during pregnancy. The present study provides further evidence of a specific maternal polymorphism that may affect the risk for ASD with exposure to prenatal stress. Autism Res 2016, 9: 1151–1160. © 2016 International Society for Autism Research, Wiley Periodicals, Inc.