"The heartless and murderous Saudis locked up the injured with the dead in containers, instead of providing medical treatment and helping them or at least quenching their thirst. They murdered them," Ayatollah Ali Khamenei said in a statement on his website marking the anniversary of the disaster.
He offered no evidence to support the allegations.
Saudi Arabia's Crown Prince and Interior Minister Mohammed bin Nayef said Iran was attempting to "politicise" the hajj.
In comments published later today by the Saudi Press Agency, he said Iran had decided not to send its citizens to the pilgrimage this year for domestic purposes.
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The September 2015 stampede and crush of pilgrims killed at least 2,426 people, according to an Associated Press count. Tehran has said 464 of the dead were Iranian and blamed the catastrophe on Saudi mismanagement of the annual pilgrimage.
Khamenei has also blamed Saudi Arabia for an earlier crane collapse in Mecca that killed 111 people, and said the kingdom's rulers had "reduced the hajj to a religious-tourist trip" while accusing Iran of "politicizing" the pilgrimage. A top Saudi official said Khamenei's accusations reflect "a new low".
"Saudi Arabia stands ready to serve the pilgrims and ensure their safety and comfort," Alyas told The Associated Press.
Iranian Foreign Ministry spokesman Bahram Ghasemi said the ministry has formed a committee to investigate the issue and pursue it in international forums, without elaborating.
The hajj stampede caused a new flare-up in tensions between Iran and Saudi Arabia, regional rivals that back opposite sides in the civil wars in Syria and Yemen.
Saudi authorities have not released the findings of their investigation into the hajj disaster. Preliminary statements suggested the crush was caused when at least two large crowds intersected.