Shirley-Anne Somerville, who was director of communities at Yes Scotland, is instead taking up a new role with the SNP.
Her departure has fuelled concerns about the lack of women in key positions at the organisation.
As communities chief, Somerville - previously a Lothians MSP who lost her seat in 2011 - had the task of establishing a network of pro-independence activists. She was believed to have built a solid base and was respected by the different elements of the independence coalition.
While Somerville was absent, Yes Scotland brought in new faces to the communities team. One insider said the division had "changed", and the director's post no longer existed. This appears to confirm the view inside Yes Scotland that too many director-level posts were created in the early days.
However, concerns have also been expressed about the number of senior female employees leaving Yes Scotland. Of the four directors unveiled at the beginning of Yes Scotland, three were women - communications chief Susan Stewart, operations director Jacqueline Caldwell and Somerville. Now all three have gone.
One source said the departures did not bode well for Yes Scotland trying to bridge the gender gap in support for independence, with polls showing more men than women backing separation.
A source at Better Together, which campaigns for Scotland to remain part of the UK, said: "The SNP campaign is constantly telling us about this army of grass-roots activists they are building. It must be hard to build an army when the generals keep resigning."