Houses at Auvers II was hung in the Picnic Cafe on Friday to launch the Open for Art weekend festival.
David Brooks, from Toronto, Canada, has been a van Gogh specialist for 18 years and claims it is not genuine.
Owner Mark Lawrence insists it is a real van Gogh, but admits it has not yet been authenticated.
Mr Brooks, who the Van Gogh Museum said "owns the largest van Gogh website in the world", told the BBC it was not a known work by the artist.
"To me it screams 'not van Gogh'," he said, adding: "It's not well executed, the style is quite poor."
The Van Gogh Museum, in Amsterdam, is the only place that can officially authenticate a van Gogh work.
Mr Lawrence said he had been working with the museum to get the painting researched since the beginning of the year.
The museum said it had received an email from Mr Lawrence on Monday, and had advised him to send a request for research along with a photograph of the painting.
It added an investigation could take weeks or sometimes months, depending on the type of inquiry required.
Mr Lawrence said: "We are doing everything that would normally be done to get it authenticated."
In relation to Mr Brooks' comments, he said: "It can't be fully authenticated simply from looking at a photograph, it has to be tested."
He also said it was possible the painting had areas of over-paint from restoration which "covers up some of the details".
"There's a lot of heavy cracking on our painting too which is commonly found in van Gogh work particularly ones he painted thickly, and it also has a protective thick varnish on it," he added.
Mr Lawrence previously said he inherited the painting from his grandfather who died in 1980, along with 200 other paintings.
The 27-year-old director of the web-based Reading Gallery is looking for a permanent home for his collection, which is currently in storage and is said to include works by Rembrandt, Picasso, and Dali.
He said House at Auvers II was bought in the 1920s by his great-grandfather for 300 francs in Paris, and added it was not known to the art world because it had been in his family since before van Gogh's works were first catalogued in the 1930s.
Doubt has been raised over the authenticity of a painting purported to be by Vincent van Gogh which was displayed in a Reading cafe.