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Recent studies have demonstrated a great potential for the use of molecular biology in Reticulitermes species identification and to study other aspects of termite biology. This study utilized micro-morphological identification and microsatellite Simple Sequence Length Polymorphic (SSLP) DNA genotyping to characterize subterranean termites collected from 73 locations across the state of Nebraska. Morphological characters of soldier termites from 30/73 collection sites revealed the presence of two species, Reticulitermes flavipes (Kollar) and R. tibialis Banks. Morphology of the gular plate ratio was used for species differentiation. Deoxyribonucelic acid (DNA) was extracted from termite workers (from all samples) and used as a template for the polymerase chain reaction (PCR) to amplify nine different genomic microsatellite loci. PCR products were resolved byagarose gel electrophoresis and visualized with ethidium bromide staining. Comparison of micro-morphological characters to the genotyping data for workers from the 30 soldier sample sites revealed that PCR-based genotyping reliably separated the samples into genotypic groups that corresponded with morphological characterization. Genotypic analysis of workers from all 73 sample sites revealed genotypic differences between the two species at four loci. Three different genotypic groups were identified as R. flavipes, R. tibialis, and mixed genotypes. Data from both characterizations were used to construct termite distribution maps based on collection sites for the two species, enabling possible future research to focus on zones of cohabitation between the species.