In support of the National Program for Chagas Disease Control, we conducted a cross-sectional study to estimate the seroprevalence rate of Trypanosoma cruzi infection across the five Departments (Chiquimula, Jalapa, Zacapa, Jutiapa, and Santa Rosa) that are believed to comprise the entire principal endemic area in Guatemala. Also, so that the results could be used to identify areas of active transmission, we conducted the survey in school-aged children. We used an experimental enzyme-linked immunosorbent assay with blood samples obtained by finger prick to estimate the seroprevalence of T. cruzi. This assay has been previously tested and showed good sensitivity and specificity. Overall, the seropositivity rate for T. cruzi infection was 5.28% (235 of 4,450). Of 173 communities evaluated, 35 (20.23%) had a seropositive rate ranging from 10% to 45%. A number of parameters, including but not limited to living conditions, were examined for possible association with seropositivity. While there are several associations, the strongest association with seropositivity is living in a house with a thatch roof. The survey results will permit the Ministry of Health to stratify T. cruzi-endemic communities, enabling local health authorities to efficiently focus on vector control operations.