An emergency order remains in place prohibiting the movement of bees in the state after the deadly varroa mite was found at hives near the Port of Newcastle last week.
A second detection was made 10 kilometres away in a commercial hive.
Acting chief executive of the Australian Honey Bee Industry council Danny Le Feuvre says authorities are working to identify and destroy all of the hives within the 10km zone.
"In the 10km zone the agreed emergency response is to eradicate all ... honey bees," he said, estimating the elimination to include about 300 hives.
Mr Le Feuvre said while Sunday's emergency order is valid for six months and stops people in NSW from moving their bees, he did not expect it to be in place that long.
"Even when that is lifted, hopefully within a week or two weeks, there will still be some restrictions in the Newcastle area," he told AAP.
The order is impacting around 270,000 hives.
"Whilst there's a standstill no one can move bees in the whole state, there are really big and significant fines in place, even jail time," he said.
"We are coming up to almond season so we're making sure we go hard early to try and establish the perimeter; make sure we have got it contained before we hit almond pollination."
Hundreds of people are responding to the bee emergency at a state control centre, and on the ground as part of additional efforts to locate and surveil hives.
Urgent tests are underway to determine whether the mites found in NSW have deformed wing virus.
The Commonwealth Scientific and Industrial Research Organisation's (CSIRO) expert on honeybee pathogens, John Roberts, told AAP the mite and the virus often come together - and when they do, they are a dangerous duo.
The mites feed on the blood of adult and larval bees and over time can weaken and kill colonies.
If the mites have the virus they can pass it to bees while enjoying their blood meals.
Infected bees end up with deformed wings, abdomens and other problems.
"If it's just the feeding damage, and not the virus damage as well, it's much lower impact than in combination, when they are acting together," Dr Roberts said.
"It will be important to keep monitoring (for the virus) at the same time as monitoring the mite, because it is as significant a threat as the mite."
The Elizabeth Macarthur Agricultural Institute is doing the testing with support from the CSIRO.
Ana Martin, who runs Amber Drop Honey on the mid north coast, told AAP it is a worrying time for the commerical bee industry.
"At the moment we don't know ... the extent of how far the hives have been infected," the beekeeper told AAP.
"As hard as it is to destroy hives ... we know the destruction the mite can cause.
"If there is any possible way that we can stop it that will always be the argument."
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