Aim: Toxoplasma, rubella and cytomegalovirus (CMV) are among the most common intrauterine infection agents associated with congenital anomalies in the world. In this study, it was aimed to determine the seroprevalence of toxoplasma, rubella and CMV in pregnant women in Istanbul by ELISA screening tests, to evaluate them according to three different age groups and to compare them with the literature. Material and Methods: In 5485 serum samples of pregnant women, IgM and IgG antibodies against toxoplasma, rubella and CMV were investigated and total of 16496 tests were evaluated retrospectively. Samples were tested on the Architect i2000SR (Abbott Diagnostics, USA) using kits based on the chemiluminescence microparticle enzyme immunassay principle (Architect, USA). Results: Toxoplasma, rubella and CMV IgM positivity rates were 0.6%, 0.3% and 2.7%, respectively, and 23%, 89.1% and 98.7% for IgG, respectively. The toxoplasma IgG seropositivity rate was highest in the 36-49 age group (p =0.012). The toxoplasma IgM seropositivity rate was the lowest in the 26-35 age group (0.5%). The lowest rubella and CMV IgG seropositivity were found in 36-49 (87.8%) and 26-35 (98.4%) age groups. Conclusion: In pregnant women, serological screening tests seem to be important for monitoring and informing high risk groups, especially seronegative, due to the low seroprevalence of toxoplasma. However, routine screening of rubella and CMV in the pregnant population should be questioned due to the high immunity rates.