The　application　of　the　metal-organic　framework　(MOF)　MIL-53(Fe)　as　a　novel　stationary　phase　for　reverse-phase　high　performance　liquid　chromatography　(HPLC)　separation　of　positional　isomers　is　described　for　the　first　time.　Under　the　optimized　conditions,　baseline　separation　of　xylene,　dichlorobenzene,　chlorotoluene　and　nitroaniline　isomers　was　achieved　on　the　MIL-53(Fe)　packed　column　within　a　short　time.　The　retention　mechanisms　of　these　isomers　on　the　MIL-53(Fe)　packed　column　were　discussed　in　detail,　and　a　typical　reverse-phase　behavior　can　describe　the　results.　The　thermodynamic　characteristics　of　the　HPLC　separation　of　positional　isomers　were　also　evaluated.　It　was　confirmed　that　the　HPLC　separation　of　positional　isomers　was　controlled　by　the　Gibbs　free　energy　change　(Delta　G).　In　addition,　reverse-phase　HPLC　separation　of　the　tested　isomers　was　also　carried　on　MIL-53(Al,　Cr)　packed　columns　to　investigate　the　influence　of　the　metal　centre　of　the　MIL-53　framework　on　their　chromatographic　behaviors.　The　results　showed　that　the　MIL-53(Fe)　packed　column　exhibited　better　separation　performance　than　the　MIL-53(Al,　Cr)　packed　columns.　Moreover,　the　MIL-53(Fe)　packed　column　also　gave　efficient　HPLC　separation　of　alkylbenzene　and　naphthalene,　aniline　compounds　and　polycyclic　aromatic　hydrocarbons　(PAHs).　These　successful　applications　suggest　the　potential　of　MIL-53(Fe)　as　a　novel　stationary　phase　for　efficient　reverse-phase　HPLC　separation.