Intraspecific genetic variation among Triatoma dimidiata (Hemiptera: Reduviidae) from seven Costa Rican populations and from different domestic, peridomestic, and sylvatic ecotopes were analyzed. The complete nucleotide sequence of the nuclear ribosomal DNA internal transcribed spacer (ITS-2) and partial sequences of the cytochrome B (Cyt b) gene and the large ribosomal subunit RNA (16S) of mitochondrial DNA (mtDNA) were analyzed and compared. All ITS-2 sequences analyzed were identical and correspond to the haplotype T.dim-H1, the most common haplotype in Central American populations. Sequences of mtDNA revealed a 10.17% of polymorphism in Cyt b and 2.39% in 16S, suggesting that the Cyt b fragment is a useful marker to describe the genetic structure of populations, even at habitat-related level. The analyses of the 18 new combined T. dimidiata haplotypes (Cytb/16S/ITS-2) showed that the two main geographical locations and populations studied are genetically structured showing different haplotype profiling. Only one combined haplotype was shared in the studied areas (Cytb.d/16S.a). Seven haplotypes exclusive for domestic/peridomestic populations, five for sylvatic, and six shared haplotypes for both habitat-related ecotopes are described. Although the relationship between the habitat and the haplotype profiling is less clear, there are different patterns of haplotype distribution in each geographic area between the two habitat-related ecotopes studied (domestic/peridomestic and sylvatic), some of them reflected in the phylogenetic relationships analyzed. The intraspecific variability detected may underlie the known plasticity of T. dimidiata, an important vector for Chagas disease transmission, suggesting that this species must be continuously monitored.