Glenys Dickson left the Gore office just over two weeks ago because of an "employment issue''.
Her replacement, Bernadette Hunt, of Gore, starts on Monday.
The second resignation, Barbara Swan, in the Queenstown office, is yet to take effect; it is understood she leaves at the end of next month.
Mrs Dickson's departure from the Gore office after 18 years was a loss, National Party Gore branch secretary Maeva Smith said.
Asked if she had confidence in Mr Barclay, Mrs Smith said: "I'd prefer not to answer that''.
"There is an employment issue, but we can't talk about it at the moment.
"Hopefully, this can be dealt with and it can be tidied up.
"The electorate [office] needs to get on with ... looking after the constituents. We need to be able to move on and get things sorted, but at the moment it hasn't happened.''
The issue had "blown up in the last three weeks'', Mrs Smith said, declining to elaborate further.
Mrs Smith said Mrs Dickson built a good list of contacts and relationships and had worked hard for constituents.
Contacted yesterday, Mrs Dickson said she could not comment on the reasons for her departure.
Paying tribute to her old boss and Mr Barclay's predecessor, Deputy Prime Minister Bill English, who is now a list MP, she said: "He's a man of integrity.''
Mrs Dickson told the Otago Daily Times she would miss the job, as she enjoyed getting "good outcomes'' for constituents.
"You make a lot of good connections and you get to know a lot of people, so you get things solved quickly.''
"I've had an awful lot of phone calls and support [since resigning].''
When contacted, Mr Barclay confirmed the "employment matter'' but declined to comment; the ODT only asked about Mrs Dickson's departure, as Ms Swan's resignation emerged later yesterday when Mr Barclay was unavailable.
He told Fairfax Ms Swan resigned to travel and spend time with family and had given plenty of notice.
Ms Swan could not be contacted.
A spokeswoman for the Parliamentary Service, which employs electorate secretaries, declined to comment.
Mr Barclay (25), Parliament's youngest MP, was elected in 2014.
