Immunological diagnostic methods for Trypanosoma cruzi depend specifically on the presence of antibodies and parasitological methods lack sensitivity during the chronic and “indeterminate” stages of the disease. This study performed a serological survey of 1,033 subjects from 52 rural communities in 12 of the 18 municipalities in the state of Querétaro, Mexico. We detected anti-T. cruzi antibodies using the following tests: indirect haemagglutination assay (IHA), indirect immunofluorescence assay (IFA), ELISA and recombinant ELISA (rELISA). We also performed Western blot (WB) analysis using iron superoxide dismutase (FeSOD), a detoxifying enzyme excreted by the parasite, as the antigen. Positive test results were distributed as follows: ELISA 8%, rELISA 6.2%, IFA and IHA 5.4% in both cases and FeSOD 8%. A comparative study of the five tests was undertaken. Sensitivity levels, specificity, positive and negative predictive values, concordance percentage and kappa index were considered. Living with animals, trips to other communities, gender, age, type of housing and symptomatology at the time of the survey were statistically analysed using SPSS software v.11.5. Detection of the FeSOD enzyme that was secreted by the parasite and used as an antigenic fraction in WBs showed a 100% correlation with traditional ELISA tests.