Dirofilaria immitis infection is an important parastic disease in many mammals, especially canids, but has not been reported in bird hosts. Filarial worms were isolated from the lumen of the right atrium of the heart and the connective tissue of the lung of a captive female Humboldt penguin, Spheniscus humboldti, that died at a zoo in Japan. One of these worms was observed morphologically and identified as D. immitis by features such as 4 pairs of cephalic papillae, 1 pair of cervical papillae, esophagus divided into 2 regions, 4 pairs of pre-anal papillae, 5 pairs of post-anal papillae, and unequal spicules. In addition, the partial DNA sequence (234 bp) of mitochondrial CO | gene of the filarial worm was identical to that of D. immitis. This is the first report of D. immitis infection in a bird.