African swine fever (ASF) poses a major threat to swine health and welfare worldwide. After several European countries have reported cases of ASF, Greece confirmed officially the first positive case on 5 February 2020. The owner of a backyard farm in Nikoklia, a village in Serres regional unit, Central Macedonia, reported a loss of appetite, weakness, dyspnea, and the sudden death of 6 domestic pigs. Necropsy was performed in one gilt and findings were compatible with acute to subacute septicemic disease. Predominantly, hyperemic enlargement of spleen and lymph node enlargement and/or hemorrhage were observed. Description of vague clinical signs by the farmer suggested a limited resemblance to ASF-acute infection. However, the disease could not be ruled out once septicemic condition including splenomegaly, was diagnosed macroscopically at necropsy. In addition, considering the farm’s location near to ASF protection zones, a further diagnostic investigation followed. Confirmation of the disease was obtained using a series of diagnostic tests on several tissue samples. Further clinical, molecular, and epidemiologic evaluation of the farm was performed. According to the contingency plan, authorities euthanized all 31 pigs on the farm, whilst blood testing revealed ASF virus infection. Further emergency measures were implemented to contain the spread of the disease.