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Bean leaf beetle, Cerotoma trifurcata (Forster) (Coleoptera: Chrysomelidae), is a common pest of soybean in the Midwest United States. However, there are currently no reports on the genetic variability of C. trifurcata. This study examined 15–30 individuals from 25 sample locations to estimate genetic variability and gene flow within and among C. trifurcata from across the Midwest. Amplified fragment length polymorphism generated 175 markers for analyses. Results from analysis of molecular variance (AMOVA) indicated that the majority of genetic variation was from within samples; only a small amount of the total variation was attributed to the variation among the samples. The GST for the entire C. trifurcata population indicated that the majority of genetic variation was found within the samples, further supporting the AMOVA results. The estimated average gene flow among the C. trifurcata samples was 1.83. The Mantel test revealed no indication of correlation between geographical and genetic distance for all the C. trifurcata samples. These findings show that C. trifurcata in the Midwest are genetically heterogeneous and part of a large, interbreeding population.