The unity of Muslims is a "definite duty and a Qur'anic order", Ayatollah said adding some regional governments' decision to normalize ties with Israel is a "great sin and mistake".
The United Arab Emirates, Bahrain, Sudan, and Morocco agreed to normalise ties with Israel in 2020, as Washington under the administration of then-US President Donald Trump made Arab-Israeli rapprochement a foreign policy priority.
"These governments must return from this path which is against the Islamic unity and make amends," Iran leader told a gathering of Muslim scholars at a Unity Conference in Tehran.
The annual Unity Week conference held on the birth anniversary of Prophet Muhammad (Pbuh) celebrated separately on 12th and 17th Rabiul Awwal of Hijra calendar by Sunni and Shia Muslims brings together scholars, intellectuals and clergy from both sects from around the world to Tehran annually. Iranian leader told the gathering that they have a duty to explain and promote the "comprehensiveness of Islam in all matters of life".
Ayatollah Khamenei said the reason for his repeated emphasis on the issue of unity is the big schism which exists among religions, with the enemies striving seriously to widen it.
"You see that the issue of being a Sunni or a Shia has entered the American political literature for several years, while they are against the quintessence of Islam and hostile to it," he said.
"The recent unfortunate and distressing explosions at mosques targeting Muslim worshipers in Afghanistan are among such incidents that were carried out by Daesh," the Leader said.
"Who is Daesh? Daesh is the same outfit that the Americans -- the same Democratic group of the United States -- explicitly said 'we created', even though they are not saying it anymore and deny it."
Ayatollah Khameni said one of the ways to prevent such incidents is for the current Afghan officials to attend Shia centers and mosques and encourage Sunni Muslims to participate in inter-religious congregations.
"It is not possible to achieve the important goal of creating a new Islamic civilization without the unity of Shias and Sunnis," Iran leader said.
Ayatollah Khamenei called Islam a comprehensive religion and the birth of Prophet Muhammad (Pbuh) a very great event and the beginning of a new era of divine blessings in human life.
Shortly after Ayatollah's speech, Iran's top security official, Ali Shamkhani, pledged to inflict many "billions of dollars" worth of damage in a "shocking response" if Israel strikes Tehran's nuclear programme.
The tweet by the secretary of Iran's Supreme National Security Council came in response to Israeli media reports that 5 billion shekels ($1.5bn) had been approved to prepare the military for a potential strike on Iran's nuclear programme.
Iran has repeatedly accused Israel of being behind acts of sabotage targeting its nuclear facilities.