The company opened at the Macdonald Aviemore hotel in the Highlands in October, but a statement on the resort's website says it closed on 18 January.
Chick-fil-A has faced criticism over past donations to organisations with alleged anti-LGBT+ policies, but the restaurant group says it has no social or political agenda.
Over 1200 people signed a petition calling for the closure of its Scottish site.
A Chick-fil-A representative told BigHospitality the restaurant was a “temporary pilot licensed location”.
The spokesperson said: “It has been our pleasure to serve guests at this pilot restaurant for the past several months, and we are grateful to Macdonald Hotels for allowing us the opportunity to learn from each and every customer.
“These insights will help us immensely as we look to having a permanent location in the UK in the future.”
The closure leaves the group with only one UK location, in Reading’s Oracle shopping centre, which is to close this spring at the end of its six-month lease.
Controversy has surrounded Chick-fil-A for years; in 2012 the group faced a boycott after its CEO made comments opposing same-sex marriage.
Last year the chain said it was ceasing donations to several companies with a history of anti-LGBT+ policies, after fulfilling its 'multi-year commitments'.
In an interview with BisNow Chick-fil-A president and chief operating officer Tim Tassopoulos said the company "needed to be clear" about its message as it expanded in to new markets.
Macdonald Hotels and Resorts declined to comment.