"The outbreak at two smallholdings was registered on Saturday, samples were taken and sent for analysis, they came back positive for bird flu," the ministry said.
All 79 birds on the smallholding have been culled. A quarantine zone has been introduced in the Tselinsky district near the site of the first case of the deadly virus.
The first bird flu outbreak was discovered in late November at the Gulyai-Borisovskaya poultry farm in the Rostov Region. The farm's entire population of 500,000 chickens was culled. Later a bird flu outbreak was registered at a smallholding close to the farm.
Another outbreak was then discovered at a farm in the Tselinsky district.
The Rostov Region is particularly vulnerable to bird flu as part of the Krasnodar Territory, which is on a route taken by migrating birds in winter. In September, the region was hit by the H5N1 strain and 230,000 birds were culled.
In February, dead poultry with traces of the lethal virus were found in Moscow, eight nearby areas. All cases were traced to a single market in southwest Moscow.
The virus, which was first isolated in humans in 1997, has been spreading rapidly, resulting in the deaths and culling of millions of birds, and a human death toll of over 200.
Although the virus has been restricted to birds, many scientists fear that it could mutate into a form transmissible between humans, unleashing a catastrophic global pandemic similar to the Spanish flu outbreak of 1918 that killed millions around the world.