The announcements came after observers from the Organization for Security and Cooperation in Europe reported a significant rise in attacks on the frontline in parts of eastern Ukraine controlled by pro-Russian rebels.
The two sides accused each other Saturday morning of fresh attacks.
"I urge my fellow citizens who are in the reserves to come to military conscription offices. Today I signed a decree on general mobilization," Denis Pushilin, the leader of the so-called Donetsk People's Republic, said in a video statement.
The leader of the Lugansk separatist region, Leonid Pasechnik, meanwhile published a decree saying the measure in his region was signed to prepare for "repelling aggression".
Western leaders have for weeks raised the alarm over a buildup of Moscow's army around Ukraine with Washington warning that an imminent attack could take place.
Pushilin claimed his region's forces had prevented attacks he said were planned by Ukraine security services, and that the Ukrainian army had continued attacks.
"Together, we will achieve for all of us the victory we desire and need. We will protect Donbas and all Russian people," Pushilin added, using the term for eastern Ukraine.
Kyiv has repeatedly denied any plans to regain control of separatist-held areas using force or of the Crimean peninsula, which was annexed by Moscow in 2014.
More than 14,000 people have been killed in fighting between Ukraine's army and Moscow-supported separatists since fighting broke out in 2014.