Cochliomyia hominivorax (Coquerel) is a myiasis fly that causes economic losses to livestock farmers in warmer American regions. Previous studies of this pest had found population structure at north and south of the Amazon Basin, which was considered to be a barrier to dispersal. The present study analyzed three mitochondrial DNA (mtDNA) markers and eight nuclear microsatellite loci to investigate for the first time the genetic diversity and population structure across the Brazilian Amazon region (Amazonia). Both mtDNA and microsatellite data supported the existence of much diversity and significant population structure among nine regional populations of C. hominivorax, which was found to be surprisingly common in Amazonia. Forty-six mtDNA haplotypes were identified, of which 39 were novel and seven had previously been found only at south of Amazonia. 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