Vigorously　developing　new　stationary　phases　to　meet　the　requirements　for　the　separation　of　positional　isomers　that　have　similar　physicochemical　properties　is　still　an　urgent　topic　in　separation　science.　Herein,　a　single-crystalline　covalent　organic　framework　(COF-300)　packed　column　for　the　separation　of　positional　isomers　in　high-performance　liquid　chromatography　(HPLC)　was　reported　for　the　first　time.　Benefitting　from　its　regular　shape,　excellent　chemical　and　thermal　stability,　microporous　feature,　and　strong　hydrophobicity　of　single-crystalline　COF-300,　the　single-crystalline　COF-300-packed　column　showed　excellent　resolution　for　the　separation　of　positional　isomers,　including　nitroaniline,　dichlorobenzene,　dibromobenzene,　diiodobenzene,　diethylbenzene,　chloronitrobenzene,　bromonitrobenzene,　and　iodonitrobenzene　isomers,　which　cannot　be　all　separated　on　commercial　columns　and　a　polycrystalline　COF-300-packed　column.　Especially,　the　resolution　values　for　m-/p-diiodobenzene　and　o-/m-diiodobenzene　were　4.45　and　2.53.　Moreover,　the　alkylbenzene,　monosubstituted　aromatics,　polycyclic　aromatic　hydrocarbons,　and　the　mixture　of　ethylbenzene　and　styrene　were　also　baseline　separated　on　the　single-crystalline　COF-300-packed　column.　This　successful　application　not　only　confirmed　the　great　potential　of　single-crystalline　COFs　in　HPLC　separation　of　positional　isomers　but　also　pioneered　the　utilization　of　single-crystalline　COFs　in　separation　science.　©　2022　American　Chemical　Society.　All　rights　reserved.