Background: Hepatitis A virus (HAV) is highly endemic in the Amazon. However, few data are available regarding HAV genotypes circulating in this region of the world. The aim of the present study was to characterize genetically HAV isolates circulating in the Brazilian part of the Amazon. Methods: Blood samples were collected from 134 IgM anti-HAV positive patients (sporadic cases). Viral RNA was extracted and the virion protein (VP)1/2A junction region of the HAV genome was successfully amplified by reverse transcription–polymerase chain reaction for 81 samples (60.4%). Nucleotide sequences (210 bp) of the 81 isolates were determined. Results: All HAV samples were from genotype I, with 78 (96.3%) belonging to subgenotype IA and three (3.7%) to subgenotype IB. By phylogenetic analysis, it was shown that 72/78 of the subgenotype IA isolates formed a cluster separated from the other South American HAV isolates previously characterized. Conclusion: The present study provides valuable new data on the genetic relatedness of HAV from the Amazon. Subgenotype IB circulates in the Brazilian Amazon but the predominant genotype is 1A, similar to what occurs in most South American countries.