Agistemus lobatus Ehara, 1964 and A. terminalis (Quayle, 1912) (Stigmaeidae) are cosmopolitan predatory mites and appear on various plants in Japan. They are so close morphologically and can be mainly separated based on the lengths of dorsal setae sci and c2. The present study aimed to redescribe these two species for detailed morphological variations and generate sequences of partial mitochondrial cytochrome c oxidase subunit I (COI) as DNA barcode data. Our results with DNA barcoding and morphology well separated A. lobatus from the close species A. terminalis.