That said, the department stands behind Secretary of State John Kerry's assessment that the number of extremists within the rebel forces remains at less than 20 percent, Little told reporters at the Pentagon.
DOD and State Department arguments on the level of extremist influence among the rebels run contrary to recent reports Islamic militants make up nearly 50 percent of the Syrian opposition.
Jabhat al-Nusra fighters, as well as militants from al Qaeda's Iraqi faction have been fighting alongside anti-government rebels battling forces loyal to Syrian President Bashar Assad in the country's civil war.
But senior U.S. military officials say the number of hardcore Islamist groups is steadily increasing as the war drags on into its third year.
Pentagon officials estimate that extreme Islamist groups now constitute “more than 50 percent” of the rebel force, “and it’s growing by the day," according to NBC News.
As the Pentagon downplays the rise of Islamic militants in Syria, Washington has begun directly supplying weapons and equipment to the rebels.
The weapon supplies, consisting mostly of small arms, ammunition and anti-tank weapons, are being coordinated by the CIA and limited to vetted portions of Syria's rebels.
President Obama approved the weapons program back in July, giving the CIA the green light to begin arming Syrian rebels from clandestine bases in Turkey and Jordan, according to reports at the time.
Top Pentagon leaders, including Defense Secretary Chuck Hagel and Chairman of the Joint Chiefs of Staff Gen. Martin Dempsey, have pushed back against arming Syria's rebels over concerns of growing extremist influence in the country.
U.S. weapons are trickling into Syria just as White House is pursuing a diplomatic solution to Washington's current stand off with the Assad regime.
American forces are poised to begin military strikes against Assad's forces, in retaliation for alleged chemical weapons attacks by the regime against the rebels.
But those strikes are now on indefinite hold, as U.S. diplomats pursue a Russian plan to force Assad to hand over his chemical weapon stockpiles to international control.
Syrian officials have reportedly agreed to the basic tenets of the plan, which would have United Nations officials take control of the country's chemical arsenal.
Kerry is meeting with Russian Foreign Minister Sergei Lavrov this week in Geneva to hammer out the details of the Russian disarmament plan.