Objectives: To study whether advanced maternal age and maternal grandmother age are associated with increased risk of Down syndrome siblings in a group of Jordanian families. Methods: This study was conducted on 127 confirmed Down syndrome cases with the age range of 18 weeks gestation to 15 years old, which were referred between the period of 2005-2008 for cytogenetic analysis at the Cytogentics section, Princess Iman Research and Laboratory Sciences Center/King Hussein Medical Center. Maternal and grandmaternal mother ages were obtained directly from the study group when the samples were collected from siblings. The maternal age ranged between 19-45 years while the maternal grandmother’s age ranged between 15-49 years. One hundred healthy families were randomly recruited from the hospital staff as a control group. Logistic regression was used for statistical analysis. Results: One hundred seventeen down syndrome cases had free trisomy 21, 7 with translocation, 2 mosaic and one with double aneuploidy (47,XXY, +21). Fifteen cases were diagnosed prenatally while 112 were diagnosed postnatally. The effect of maternal age and maternal grandmother age were found to be significant using logistic regression statistics (P = 0.001; OR= 2.816; 95% CI, 1.48-5.33) for the mother’s age and (P = 0.001; OR= 2.902; 95%CI, 1.521-5.53) for the grandmother’s age. Conclusion: Advanced maternal and maternal grandmother ages are risk factors for Down syndrome. More studies and investigations are needed for better understanding of the biological factors responsible for the proper meiotic segregation of germ cells during the fetal development of the embryo in advanced maternal and grandmother’s age.