The population genetics of Triatoma dimidiata haplogroups was analyzed at landscape and sub-regional scales in Chiapas and regional level across the Mexican Neotropics, and phylogeography of the complex was re-analyzed across its complete geographic range. Two contiguous fragments of the ND4 gene were analyzed due to bias from differential haplogroup specificity using a previously designed sequence. At both landscape (anthropic modification gradient) and regional (demographic, fragmentation, biogeographic, climate) scales, lowest T. dimidiata genetic diversity occurs where there is greatest historical anthropic modification, and where T. cruzi infection prevalence is significantly highest. Trypanosoma cruzi prevalence was significantly higher than expected in haplogroups 1 and 3, while lower than expected in haplogroup 2. There was also a significant difference of DTUI and DTUVI infection frequencies in both haplogroups 1 and 3, while no difference of either in haplogroup 2. All haplogroups from the Mexican Neotropics had moderate to high haplotype diversity, while greatest genetic differentiation was between haplogroups 1 and 3 (above FST = 0.868, p < 0.0001). Divergence of the complex from the MRCA was estimated between 0.97 MYA (95% HPD interval = 0.55–1.53 MYA) and 0.85 MYA (95% HPD interval = 0.42–1.5 MYA) for ND4A and both concatenated fragments, respectively, with primary divergence from the MRCA of haplogroups 2 and 3. Effective population size for Mexican haplogroups 1 and 2 increased between 0.02 and 0.03 MYA. This study supports previous ecological niche evidence for the complex´s origin surrounding the Tehuantepec Isthmus, and provides evidence for recent divergence of three primary dimidiata haplogroups, with differential T. cruzi infection frequency and DTU specificity, important components of vector capacity. Triatoma dimidiata is one of the broadest distributed triatomine species´ complexes transmitting Trypanosoma cruzi. In Mexico, three haplogroups of the T. dimidiata complex have been reported and all are primary vectors of Chagas disease south of the Tehuantepec Isthmus. Given their epidemiological importance, the question arises whether haplogroups have similar genetic diversity in domestic/modified landscapes, as well as infection characteristics and parasite DTU associations, key components of vector capacity. The aim of the present study was to analyze Triatoma dimidiata population genetics across landscapes, sub-regional, regional, and global Neotropical realm scales, using two contiguous fragments of the ND4 gene. Our results support previous evidence for the complex´s origin surrounding the Tehuantepec Isthmus, and provides evidence for recent divergence of three principal dimidiata haplogroups and significant secondary divergence within each. Differential T. cruzi prevalence and Discrete Typing Unit (DTU) specificity for individual haplogroups provide evidence for potential differential vector capacity within the complex in Mexico.