Trypanosoma cruzi is the causative agent of Chagas disease, a devastating parasitic disease endemic to Central and South America, Mexico, and the USA. We characterized the genetic diversity of Trypanosoma cruzi circulating in five triatomine species (Triatoma gerstaeckeri, T. lecticularia, T.indictiva, T. sanguisuga and T. recurva) collected in Texas and Southern Arizona using multilocus sequence typing (MLST) with four single-copy loci (cytochrome oxidase subunit II- NADH dehydrogensase subunit 1 region (COII-ND1), mismatch-repair class 2 (MSH2), dihydrofolate reductase-thymidylate synthase (DHFR-TS) and a nuclear gene with ID TcCLB.506529.310). All T. cruzi variants fall in two main genetic lineages: 75% of the samples corresponded to T. cruzi Discrete Typing Unit (DTU) I (TcI), and 25% to a North American specific lineage previously labelled TcIV-USA. Phylogenetic and sequence divergence analyses of our new data plus all previously published sequence data from those four loci collected in the USA, show that TcIV-USA is significantly different from any other previously defined T. cruzi DTUs. The significant level of genetic divergence between TcIV-USA and other T. cruzi DTUs should lead to an increased focus on understanding the epidemiological importance of this DTU, as well as its geographical range and pathogenicity in humans and domestic animals. Our findings further corroborate the fact that there is a high genetic diversity of the parasite in North America and emphasize the need for appropriate surveillance and vector control programs for Chagas disease in southern USA and Mexico.