Migrant workers Zaw Lin and Win Zaw Tun are on trial for the murder of 24-year-old David Miller and the rape and murder of Hannah Witheridge, 23, on Koh Tao island in September.
If found guilty they could face the death penalty. Both have pleaded not guilty.
Thai police and prosecutors say forensic evidence strongly points towards the two 22-year-old suspects with both men's DNA found on or inside the body of Witheridge.
They had asked trial judges on the nearby island of Koh Samui permission to independently retest all the police's DNA samples.
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On Friday the court ruled that they could do so.
But there was confusion about which samples left in police possession were still in a suitable state for retesting -- particularly the crucial swabs taken from Miller and Witheridge's bodies.
Lead defence lawyer Nakhon Chomphuchart told AFP that only a handful of items found near the crime scene can be reexamined, including a garden hoe -- the suspected murder weapon -- a shoe, a sock and some plastic bags.
"We wanted to be able to test more but with the other items there are no samples left (to test)," he said.
It is not clear how useful a retest on the available items will prove to either side. Earlier in the week a witness testified to removing and washing down the garden hoe when he came across it shortly after the holidaymakers' bodies were found.
"The Court order means no possible retest on DNA samples from Hannah and David's bodies," Andy Hall, a Thailand-based British rights activist who has raised money for the accused and advises their legal team, told AFP.