With the conquest of Samarkand by Shibanian Abu al-Khairi (1501 AD) at the same time as the establishment of the Safavid rule, a process began, the result of which was the religious confrontation between Transoxiana and the inner parts of Iran. This development marked the first stage of the political separation of the Transoxiana from Iran, which led to complete separation a few centuries later with the intervention of Russia. In the investigation of the underlying factors of this process, the contribution of the political leaders has often been highlighted, and now the contribution of the scholars and their fatwas in forming this separation should also be considered. The present study, with a descriptive-analytical approach and relying on the data of the sources and the results of the studies, aims to evaluate the social status and base of the scholars in Shibani's rule and to examine the effect of their performance in the political separation of the Transoxiana region from the inner parts of Iran. The results show that the scholars were the focal point of Shibanian's political-military actions by holding important judicial, administrative, and political jobs and having financial resources. They played a key role in reformulating religious concepts such as caliphate, jihad, and Dar al-Harb, as well as issuing a fatwa in giving identity to the Shibani rule and legitimizing those newcomers by the people. In addition to that, the investigation of the performance of Transoxiana Sunni scholars in justifying the Shibanians' military-political efforts against the neighboring rules shows that their actions led to the formalization of the Shibanians' territorial claims and the establishment of the political separation of Transoxiana from Iran