Background: Infection with cytomegalovirus (CMV) is highly frequent during pregnancy in human beings, which mostly results in preterm birth. Objectives: The current study aimed at comparing serological and molecular methods to determine the frequency of CMV infection in pregnant women admitted to hospitals in Golestan Province, Northern Iran. Methods: The study was conducted on 315 blood samples collected from pregnant women. After completion of the screening test questionnaire, CMV-IgG, CMV-IgM, and CMV-IgG avidity tests were performed to determine the seropositivity prevalence of CMV. Finally, after DNA extraction, the PCR technique was employed to detect the CMV genome in the samples. Results: Out of the studied women, 81.2% were positive for CMV-IgG and CMV-IgM, but only 8.2% had positive results in the molecular detection of CMV, out of which 61.9% had a history of abortion. In terms of the correlation between ethnicity and infection with CMV, 66.7% of positive samples belonged to Fars ethnic group. The relationship between ethnicity and occupational status in terms of CMV infection revealed that 85% of Fars and 29% of Turkmen housewives were positive for CMV. In terms of the relationship between ethnicity and age, 35% of pregnant women from Fars and 42% from Turkmen ethnic groups were over 30 years. Furthermore, the results showed that 71% of Fars and 42% of Turkmen pregnant women were infected with CMV during the 2nd trimester of pregnancy. Among all these variables, a significant relationship was observed only between the age of pregnant women and infection with CMV (P = 0.045). Conclusions: The current study results showed that despite the increasing frequency of CMV infection in pregnant women, seropositivity, and even higher titers of CMV-IgG have no predictive value for active CMV infection. Concerning the importance of rapid and definitive diagnosis of the disease before the emergence of manifestations, molecular techniques could be of great help as they are effective in the diagnosis of infection with smaller amounts of the pathogenic genome.