Boris Johnson is under investigation by the Parliamentary Commissioner for Standards over his £15,000 trip to Mustique.
Kathryn Stone has confirmed for the first time that she is looking into whether the PM properly declared the holiday.
Downing Street has previously said "all relevant transparency requirements" were met around the trip to Mustique, a private island in the archipelago nation of St Vincent and the Grenadines.
The PM’s official spokesman said on Monday that Mr Johnson "transparently declared the benefit in kind in the Commons register of interests" and followed the rules "throughout".
But there was confusion when the PM declared the holiday in the register of MPs’ interests last year.
In the entry, Mr Johnson said he had accepted "accommodation for a private holiday for my partner and me, value £15,000", citing Tory donor David Ross as the provider.
But a spokesman for Mr Ross, co-founder of Carphone Warehouse, denied he had paid for it.
Mr Ross has since sought to further clarify matters, agreeing it was a "benefit in kind" to the PM and Ms Symonds during their private break.
A spokesman for Mr Ross reiterated his position on Monday, saying: "Mr Ross facilitated accommodation for Mr Johnson on Mustique valued at £15,000.
"Therefore this is a benefit in kind from Mr Ross to Mr Johnson, and Mr Johnson’s declaration to the House of Commons is correct."
The PM appears on a list of nine MPs who are under investigation by the commissioner, which has been published under new Commons rules.
Before the change, the commissioner was not allowed to say whether she was inquiring into a complaint against an MP.
Reacting to the news, Labour deputy leader Angela Rayner said: "Another day, another investigation into Boris Johnson for more sleaze and dodgy dealings.
"The public have a right to know who paid for Boris Johnson’s luxury Caribbean holiday and the renovation of his flat.
"Most importantly, we need to know what these donors were promised or expected in return for their generosity.
"As we have seen over the last year, Tory donors have received a very high return on their investment in the form of government contracts.
"Boris Johnson needs to stop using the office of prime minister as an opportunity to fund his lavish lifestyle and enrich his mates."