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The US leads a coalition carrying out air strikes on so-called Islamic State in Syria and Iraq
They argue the current approach is working against the Syrian opposition and helping Mr Assad to stay in power.
It was signed by 51 mid-to-high level officials who advise on Syria issues.
It is not unusual for internal “dissent cables” to be filed through State Department channels.
However, it is rare to have this number of diplomats voice opposition to a White House position.
A State Department spokesman acknowledged receipt of the memo but declined to comment on its contents.
However, an official familiar with the letter told the BBC that it was sent “because the status quo is not sustainable”.
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The document urges a credible threat of military action against the Assad government. Otherwise, it says, Damascus will feel no pressure to negotiate with the rebels.
This reflects concerns that the collapse of a joint US-Russian peace process is benefitting the regime.
There have been violations of the ceasefire by both the opposition and the government.
But President Assad has openly defied the truce, and his forces, backed militarily by Iran and Russia, seem intent on regaining strategic territory such as the crucial city of Aleppo.
Syrian cities such as Aleppo have been left in ruins by years of war
Moscow argues that it is supporting strikes against jihadist insurgents not covered by the ceasefire.
But Secretary of State John Kerry, who has pushed opposition groups to lay down their arms, is growing increasingly frustrated as the Syrian regime continues to change facts on the ground while he calls for diplomacy.
“The United States is not going to sit there and be used as an instrument that permits a so-called ceasefire to be in place while one principal party is trying to take advantage of it to the detriment of the entire process,” he said recently.
Mr Kerry has pressed the administration for tougher action against the regime in order to force it to the negotiating table, and signatories to the dissent letter believe he’ll be sympathetic to their concerns, according to sources involved in the process.
But that’s unlikely to sway the Obama administration, which has prioritised the fight against so called Islamic State (IS) in Syria and largely stayed clear of the civil war.
President Obama is wary of being drawn into another Mid-East conflict after the messy results of US intervention in Iraq, Afghanistan and Libya.
However, his possible successor, presumptive Democratic presidential candidate Hillary Clinton, has argued for a more assertive policy in Syria, including stronger support for non-Islamist rebels.
Perhaps the letter is aimed as much at her as it is at Mr Kerry.
BBC