The agent of Chagas disease, Trypanosoma cruzi, is phylogenetically divided into two lineages, T. cruzi I (TCI) and II (TCII). TCI is found in sylvatic and domestic habitats across South America. Despite a high prevalence of TCII in domestic habitats in South America, it has been rarely found in domestic habitats in Central America and Mexico. This may be caused by limitations in detection tests previously used. A modified hemi-nested polymerase chain reaction assay was developed to improve detection of TCI and TCII mini-exon genes. This method detected TCI and II concurrently in 84% of 44 cultured isolates from Triatoma dimidiata specimens collected inside homes across the disease-endemic area in Guatemala.