Our aim was to evaluate the association of functional polymorphism of macrophage migration inhibitory factor (MIF) gene with Chagas disease. Our study includes two independent cohorts: 240 chagasic patients and 199 controls from Colombia; and 74 chagasic patients and 85 controls from Peru. The single nucleotide polymorphism (SNP) -173 G/C of MIF gene was determined using a polymerase chain reaction (PCR) system with pre-developed TaqMan assay. We observed a statistically significant difference in the distribution of -173*C allele of MIF gene between patients and controls in the Colombian cohort (OR = 1.6, 95% CI = 1.12-2.18, p = 0.006). Similar association was found in the Peruvian cohort (OR = 2.4, 95% CI = 1.31-4.38, p = 0.003). A meta-analysis of the Colombian and Peruvian cohorts demonstrated that the -173 C allele confers a risk effect in chagasic patients (pooled OR = 1.75, 95% CI = 1.30-2.33, p = 0.0002). In addition, a gene dose of the MIF -173 C allele was observed (pooled OR = 4.01, 95% CI = 1.25-12.85, p = 0.004). Our results suggest that the MIF -173G/C polymorphism confers susceptibility to Chagas disease in the populations under study.