•The complete mitochondrial (mt) genome sequences of the Ecuadorian Amphimerus were determined.•The Ecuadorian Amphimerus mt genome has similar gene content and arrangement to those of other Opisthorchiidae.•Phylogenetic analysis placed the Ecuadorian Amphimerus within Opisthorchiidae family, but distinct from Opisthorchis.•The Amphimerus mt genome provides a new resource for future systematic studies of Opisthorchiidae trematodes. Amphimerus Barker, 1911 is a liver fluke infecting several animal species and humans. Being a digenetic trematode of the Opisthorchiidae family, Amphimerus is closely related to the genera Metorchis, Clonorchis and Opisthorchis. Recently, a high prevalence of Amphimerus infection in humans, cats, and dogs had been demonstrated in a tropical Pacific region of Ecuador. Hence, we determined and characterized the entire mt genome sequences of adult liver flukes, morphologically identified as Amphimerus, collected in the endemic region of Ecuador, and examined its phylogenetic relationships with flukes in the Opisthorchiidae family using Bayesian inference (BI) based on the concatenated amino acid sequences and partial cox1 sequences. The complete mt genome sequence (15, 151 bp in length) of the Amphimerus sp. contains 35 genes, including 12 protein-coding genes (PCGs, without atp8), two rRNAs (rrnL and rrnS) and 21 tRNAs, lacking trnG. The gene content and arrangement of the Ecuadorian Amphimerus mt genome was similar to those of other trematodes in the Opisthorchiidae family. All genes in the circular mt genome of Amphimerus sp. are transcribed from the same strand in one direction, with the A + T content of 60.77%. Genetic distances between Amphimerus sp. and other genera in Opisthorchiidae were rather high, ranging from 26.86% to 28.75% at nucleotide level and 29.37%–31.12% at amino acid level. Phylogenetic analysis placed the Ecuadorian Amphimerus within the branch of Opisthorchiidae, but very distinct from Opisthorchis. Our results indicate that the liver fluke Amphimerus from Ecuador does not belong to the genus Opisthorchis, and that it should be assigned under the genus Amphimerus. The determination of the mt genome of the Ecuadorian Amphimerus provides a new genetic resource for future studies on taxonomy and molecular epidemiology of Opisthorchiidae trematodes.