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Background: Chagas disease was originally reported in Panama in 1931. Currently, the best knowledge of this zoonosis is restricted to studies done in historically endemic regions. However, little is known about the distribution and epidemiology of Chagas disease in other rural areas of the country., Methods and Findings: A cross-sectional descriptive study was carried out between May 2005 - July 2008 in four rural communities of the Santa Fe District, Veraguas Province. The study included an entomologic search to collect triatomines, bloodmeal type identification and infection rate with trypanosomes in collected vectors using a dot- blot and PCR analysis, genotyping of circulating Trypanosoma cruzi (mini-exon gene PCR analysis) and the detection of chagasic antibodies among inhabitants. The vector Rhodnius pallescens was more frequently found in La Culaca and El Pantano communities (788 specimens), where it was a sporadic household visitor. These triatomines presented darker coloration and larger sizescompared with typical specimens collected in Central Panama. Triatoma dimidiata was more common in Sabaneta de El Macho (162 specimens). In one small sub-region (El Macho), 60% of the houses were colonized by this vector. Of the examined R. pallescens, 54.7.0% (88/161) had fed on Didelphis marsupialis, and 24.6% (34/138) of T. dimidiata specimens collected inside houses were positive for human blood. R. pallescens presented an infection index with T. cruzi of 17.7% (24/136), with T. rangeli of 12.5% (17/136) and 50.7% (69/136) were mixed infections. In 117 T. dimidiata domestic specimens the infection index with T. cruzi was 21.4%. Lineage I of T. cruzi was confirmed circulating in these vectors. A T. cruzi infection seroprevalence of 2.3% (24/1,056) was found in this population., Conclusions: This is the first report of Chagas disease endemicity in Santa Fe District, and it should be considered a neglected public health problem in this area of Panama.