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Trypanosoma cruzi is endemic to the Americas where it demonstrates multiple lineages over a vast geographic range (i.e., United States to Argentina). These lineages possess divergent geographic and biologic characteristics, including variations in disease manifestations. Herein, we report the frequency of parasitemia among seropositive US blood donors and the potential association between parasite lineage and transfusion transmission.Blood donors identified as T. cruzi seropositive during screening were enrolled in follow-up studies, including hemoculture testing and a risk factor questionnaire. Positive hemocultures were expanded to obtain sufficient parasites for molecular lineage determination and analysis. Country of birth, obtained from the questionnaire, was used to predict parasite lineage in the absence of demonstrable parasitemia for infected donors.Eighteen (6.8%) of 263 seropositive donors were hemoculture positive. Among the 17 hemocultures expanded for lineage determination, TcV was identified more frequently (n = 12), compared to TcI (n = 2), TcII (n = 1), and TcVI (n = 2). When presumptive parasite lineages were compared to hemoculture results, only two of 157 (1.3%) TcI versus 13 of 38 (34.2%) TcII/TcV/TcVI non-US donors were parasitemic; three of 44 (6.8%) US donors were TcV or TcVI.Based on lineage determination for donors with parasitemia; hemoculture positivity associated with presumptive parasite lineage; and implicated donors from US, Canadian, and Spanish transfusion cases, donors from Southern South America are significantly more likely to have parasitemia and transmit infection to blood recipients (TcII, TcV, or TcVI vs. TcI). Thus, parasite lineage may be associated with risk of transfusion-transmitted T. cruzi.