Context: Tobacco increases the risk of systemic diseases, and it has adverse effects on pregnancy. However, only indirect data have been published on a possible genotoxic effect on pregnancy in humans. Objectives: To determine whether maternal smoking has a genotoxic effect on amniotic cells, expressed as an increased chromosomal instability, and to analyze whether any chromosomal regions are especially affected by exposure to tobacco. Design, Setting, and Patients: In this prospective study, amniocytes were obtained by routine amniocentesis for prenatal diagnosis from 25 controls and 25 women who smoke (≥10 cigarettes/d for ≥10 years), who were asked to fill out a smoking questionnaire concerning their smoking habits. Chromosomal instability was analyzed in blinded fashion by 2 independent observers in routine chromosome spreads. Breakpoints implicated in chromosomal abnormalities were identified by G-banding. Main Outcome Measures: Association between maternal smoking and increased chromosomal instability in amniotic fluid cells, expressed as chromosomal lesions (gaps and breaks) and structural chromosomal abnormalities. Results: Comparison of cytogenetic data between smokers and nonsmokers (controls) showed important differences for the proportion of structural chromosomal abnormalities (smokers: 12.1% [96/793]; controls: 3.5% [26/752]; P = .002) and to a lesser degree for the proportion of metaphases with chromosomal instability (smokers: 10.5% [262/2492]; controls: 8.0% [210/2637]; P = .04), and for the proportion of chromosomal lesions (smokers: 15.7% [391/2492]; controls: 10.1% [267/2637]; P = .045). Statistical analysis of the 689 breakpoints detected showed that band 11q23, which is a band commonly implicated in hematopoietic malignancies, was the chromosomal region most affected by tobacco. Conclusions: Our findings show that smoking 10 or more cigarettes per day for at least 10 years and during pregnancy is associated with increased chromosomal instability in amniocytes. Band 11q23, known to be involved in leukemogenesis, seems especially sensitive to genotoxic compounds contained in tobacco.