Objective: To determine the prevalence and risk factors associated with Chagas disease in pregnant women in an endemic area of Santander, Colombia. Methods: Cross-sectional study included 23 municipalities of Santander, Colombia. Serological IFAT and ELISA tests were undertaken to detect IgG anti- Trypanosoma cruzi. A questionnaire was conducted for assessing the risk factors of each participant. Newborns were evaluated at birth and followed up to 1 year of age to determine congenital infection. Results: An overall prevalence of 3.2% (95% CI 2.4-4.2) among 1518 pregnant women was detected. Prevalences by provinces were as follows: Guanentina: 6.0% (95% CI 4.1-8.5), García Rovira: 2.9% (95% CI: 1.5-4.8) and Comunera: 0.4% (0.4-2.3). The main risk factors identified were age >32 years old (OR: 2.1; 95% CI: 1.1-3.9); currently having a thatched roof (OR: 11.8; CI95% 2.2-63.2) and a thatched roof during childhood (OR: 3.0; 95% CI: 1.4-6.6); having below primary school education level (OR: 4.6; 95% CI: 2.2-9.5); and a history of a close contact with the vector (triatomine bugs) at least once during their lifetime (OR: 6.9; 95% CI: 3.7-12.9). No congenital cases were detected by parasitological or serological techniques. Conclusions: Prevalence of Chagas disease in pregnant women is a potential source of infection in this Colombian endemic area. The main risk factors associated with seropositivity were related to conditions favouring the contact with the vector. The results show that it is necessary to continue an active surveillance in order to offer diagnosis and treatment to mothers and their newborns in addition to screening to pregnant women from endemic areas.