The genetic variability of the coffee berry borer Hypothenemus hampei (Ferrari) (Coleoptera: Curculionidae) was evaluated in populations from Brazil using amplified fragment length polymorphism ( AFLP). DNA samples from 15 coffee sites in Brazil were evaluated. Low genetic variability was observed, but enough polymorphisms to determine the population structure of H. hampei in Brazil. The results of this investigation allowed to comprise the entire H. hampei population from Brazil into three genetic branches, with a marginal value of directional gene flow of 6% between groups one and two and the absence of migrants from groups two and three. Of the total genetic variation of the coffee berry borer in Brazil, 96% was within populations. The total population differentiation value (F ST = 0.1679) was high; however, heterogeneity was observed in the F ST values when performing pairwise comparisons of populations. The average percentage of polymorphic loci was 16.89% among all of the samples, and the highest values were observed in the Uberlândia region (36.36%), which also harbored the highest number of unique AFLP fingerprints (seven). The results of this research allow to conclude that, contrary to old reports of the introduction of coffee berry borer in Brazil (Campinas), other coffee areas such as Uberlândia and Santa Cruz de Cabralia may have been the point of entrance of this insect pest into the country. The insect individuals from this region could serve in the development of molecular markers for further ecology and biology studies of the coffee berry borer in Brazil.