Two people have also been infected and are under hospital care in Brisbane.
Queensland chief veterinary officer Dr Ron Glanville said the disease remained confined to a vet clinic at Redlands, in Brisbane, and at a property near Proserpine, in northern Queensland.
Another horse at each of these properties has tested positive for Hendra virus this week, bringing the total number of equine cases to five at Redlands and two at Proserpine.
The latest horse to fall victim at the clinic may have been exposed to infection from some of the previous cases as the time of exposure falls within the known incubation period of between 4-16 days, Dr Glanville said.
The horse was euthanized on humane grounds on July 24. Other horses at the vet clinic appear healthy and are being monitored, he said.
At Proserpine, the new positive horse underwent a post mortem examination on July 21 after exhibiting clinical signs consistent with Hendra virus infection.
Early positive results from samples taken at the autopsy were received late on July 23.
"Another horse that was ill at the same time as the autopsied horse is recovering but will be monitored and sampled to determine if it was exposed to the Hendra virus. Other horses on the property appear to be healthy."
Both properties where Hendra virus has been detected remain under quarantine, Dr Glanville said.
"Horses will not be permitted to move from these premises until it is safe for them to do so. To lift quarantine each horse on the property must have a negative blood test from a sample taken at least 16 days after the last case of infection was identified on the property.
Strict precautions are in place on the two affected properties to prevent Hendra jumping to people.
Queensland Health has been working with the Department of Primary Industries on containment.
The natural reservoir for Hendra virus is in flying foxes.
"It is unclear how the disease spreads from flying foxes [native bats] to horses but it is suspected that infected bodily fluids from flying foxes contaminate something the horse then eats or drinks," Dr Glanville said.
He said research has shown that the virus has been present in flying foxes for a very long time. It was discovered in 1994 that the virus can spill over from flying foxes to horses and from horses to people.
Authorities are urging Queensland horse owners to immediately report sick horses that rapidly develop a fever and within 48 hours become extremely sick or even die. The animals are likely to show respiratory or neurological problems.
"Sick horses showing these signs should be kept isolated from other horses and people. Try not to handle these horses at all or, at a bare minimum, until examined," Dr Glanville said.
"Avoid contact with all bodily fluids, including nasal discharge and coughing, from these horses until veterinary advice is received.
"Wash hands with soap and water after handling sick horses, or shower thoroughly if contaminated with any bodily fluids from sick horses. Personal hygiene is part of the barrier against Hendra virus and other infections."
Any person working with horses who becomes ill after contact with a sick horse should immediately contact their doctor or local public health unit.
Horse feed and watering points should be placed under cover or away from trees that are attractive to flying foxes, to avoid possible contamination of contents by flying foxes.