Abstract The objective of this study was to analyze the genetic diversity among and within populations of Brazil nut by Amplified Fragment Length Polymorphism markers. Five populations with 30 plants each of the state of Amazonas, Brazil, two populations from the Farm Aruanã (Aruanã Carolina and Aruanã Brastor), in Itacoatiara, one from the municipality of Parintins and two from the municipality of Manaus (Instituto Federal do Amazonas - IFAM and Manaus Airport) were evaluated. Four primer combinations were used and 306 polymorphic bands (93.3%) were obtained. Genetic differentiation within and among populations was tested by analysis of molecular variance. The presence of genetic divergence was greater within populations (51.88%) than among populations (48.11%) and the Fst value was 0.48. Two groups were formed by interpopulation grouping: the first consisted of populations from plantations in Aruanã Carolina, Aruanã Brastor and Manaus IFAM, and the second of natural populations from Parintins and Manaus Airport.