Abstract We examined the diversity, habitat preferences, and intraspecific variation of Reticulitermes (Isoptera: Rhinotermitidae) populations within Missouri. Approximately 600 subterranean termite colonies were sampled from paired urban and woodland environments plus home infestations throughout the state. Samples were first identified to species using morphological characters and morphometrics. In addition, a portion of ≈428 bp from the 16S rRNA from 365 samples was sequenced to confirm morphology-based species identifications and to describe intraspecific genetic variability of Reticulitermes populations in Missouri. Reticulitermes flavipes (Kollar), Reticulitermes virginicus (Banks), Reticulitermes tibialis Banks, and Reticulitermes hageni Banks were found in Missouri. Twenty-one haplotypes of R.flavipes were identified, which represents one of the highest haplotype diversities reported for this species from a single state. Moreover, nine of these 21 haplotypes have not been reported in the United States before this study. Three haplotypes of R. hageni, five haplotypes of R. tibialis, and four haplotypes of R. virginicus were identified. The most abundant haplotypes of R.flavipes and R. hageni occur in woodlands, urban environments, and home infestations, suggesting a probable local origin for most of the subterranean termite infestations occurring in Missouri.