To those who know of such things, the Inquisition is widely believed to have been formed by Malcador the Sigillite on the orders of the Emperor of Mankind at the start of the Horus Heresy. The new organisation had been created from 4 lords and administrators of the Imperium and 8 Space Marines drawn from the Loyalist Astartes and the Traitor Legions who had remained loyal to the Emperor when the rest of their Legions had turned to Chaos. In this way they had displayed an unusual purity of mind and resistance to corruption. These 8 Astartes were gathered from across the galaxy during the Heresy by Nathaniel Garro, the former Battle Captain of the 7th Great Company of the Death Guard Traitor Legion who had stayed loyal to the Emperor and left Istvaan III behind on the Frigate Eisenstein to warn the Emperor of the Warmaster Horus' treachery. The foundation of the Inquisition is shrouded in mystery and is still a much-debated subject amongst Imperial scholars.

It is generally accepted that the Inquisition only solidified into its truly modern form some time in the 32nd Millennium after the formal birth of the Imperial Cult as the state religion of the Imperium, some 800 years after the end of the Horus Heresy, although its essential components and apparatus (the Black Ships, the "Witch Hunters" of the Sisters of Silence, etc.) were active in the Imperium during the Great Crusade before the Heresy began. newly uncovered sources indicate that the Inquisition had 12 key founders, who were active before, during and after the Great Crusade. As noted above, at the start of the Heresy the Emperor had ordered his regent, Malcador the Sigillite, to gather men and women of unswerving loyalty and devotion who might seek out the hidden foes of all Mankind. Malcador presented 12 such individuals to the Emperor, 4 of them Imperial administrators of "an inquisitive nature" and 8 of them Space Marines, including Captain Garro and a few other Loyalists he had been tasked with gathering from across the galaxy who were drawn from the remaining Loyalist Astartes Legions or who had escaped their Traitor Legions after the massacre at Istvaan III. These 8 Astartes would go on to become the first of what would later be known as the Grey Knights, the Chamber Militant of the Inquisition's Ordo Malleus, the Demonhunters. There were originally only two Ordos (the Ordo Malleus and the Ordo Xenos) within the Inquisition, but a third (the Ordo Hereticus) was added after the terrible events of the Age of Apostasy in the 36th Millennium to prevent a future Reign of Blood and "Plague of Unbelief".