Ayman al-Zawahiri, who replaced Osama bin Laden five years ago, accused ISIS boss Abu Bakr al-Baghdadi of "crimes" against Islam.
He savaged the rival terror organisation in an audio recording released on Monday.
In the message, Egyptian doctor al-Zawahiri described ISIS chiefs as "officers and intelligence [personnel] of Saddam" and said anyone who "pledges allegiance to Baghdadi must know that he's a partner in all his crimes".
Criticising the deaths of fellow jihadis, he said: "They are preoccupied with the illusion of running the Caliphate over the skulls of the Mujahideen."
The angry comments come after al-Zawahiri urged rival jihad groups in warn-torn Syria to unite or risk death before slamming ISIS – also known as Daesh – as "extremists".
Tensions have flared between the two evil regimes as land and resources shrink amid sustained air strikes from Russia and coalition forces.
The groups had reached a truce, but in-fighting between high-ranking al-Qaida and ISIS members shattered relations.
Al-Zawahiri has repeatedly questioned the legitimacy of IS leader Al-Baghdadi, who declared himself leader of the caliphate in Mosul, Iraq, last year.
But despite his attacks, he reportedly said in another audio recording that the rivals should unite to fight against Assad and western forces.
He said: ""We have to want the unity of the Mujahideen in Sham [Syria] so it will be liberated from the Russians and Western Crusaders. My brothers ... the matter of unity is a matter of life or death for you."
ISIS and al-Qaida were a single entity until 2013, when Daesh militants expanded into Syria against the wishes of al-Zawahiri.
This led al-Zawahiri to last year declare war on the fellow jihadist group, saying he had "no choice" but to to destroy the competition.
He said: “We have endured a lot of harm from Abu Bakr al-Baghdadi and his brothers, and we preferred to respond with as little as possible, out of our concern to extinguish the fire of sedition.
“But Abu Bakr al-Baghdadi and his brothers did not leave us a choice, for they have demanded that all the mujahideen reject their confirmed pledges of allegiance, and to pledge allegiance to them for what they claim of a caliphate."
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