The Galapagos sea lion (Zalophus wollebaeki), an endemic and endangered pinniped, faces an increasing threat due to infectious diseases related to domestic animals. Dirofilaria immitis, the parasite responsible for canine heartworm disease, is one such threat, as canine infections on the archipelago have been documented. We used a canine heartworm antigen test kit to analyze the blood from 25 juvenile Galapagos sea lions for D. immitis. Two (8%) sea lions tested positive for D. immitis antigen. Using morphologic and genetic assessments, we evaluated 20 filarial-like worms collected from within the heart of an adult male Galapagos sea lion during a previous routine postmortem examination. The intracardiac worms were morphologically consistent with adult D. immitis, and sequence analysis of targeted PCR amplicons confirmed their identity. This is the first report of D. immitis infection in Galapagos sea lions, which could become a major health problem for these pinnipeds. Further studies are necessary to confirm the level of threat from this parasite; however, widespread adoption of routine heartworm testing, prevention, and treatment in the canine population, and the control of mosquitos, could potentially reduce the disease impact on this endangered pinniped species.