Disease can threaten the restoration of endangered species directly by substantially decreasing host survival or indirectly via incremental decreases in survival and reproduction. During a biomedical survey of reintroduced populations of the highly endangered black-footed ferret from 2002 to 2005, microfilariae discovered in the blood were putatively identified as Dirofilaria immitis, and widespread screening was initiated using a commercially available antigen-based ELISA test. A subset of animals (n=16) was screened for D. immitis using a highly sensitive PCR-based assay. Microfilariae were also molecularly and morphologically characterized. Of 198 animals at six reintroduction sites, 12% had positive results using the ELISA test. No antigen-positive animals which were screened via PCR (n=11) had positive PCR results, and all antigen-positive animals (n=24) were asymptomatic. No significant differences were found in body mass of antigen-positive (male: 1223±82 g [mean±SD], female: 726±75 g) vs. antigen-negative (male: 1,198±119 g, female: 710±53 g) individuals (P=0.4). Antigen prevalence was lower in juveniles (3%) than adults (12%; P=0.03), and higher in in situ, captive-reared individuals (33%) than wild-born individuals (10%; P=0.005). Morphologic analysis of microfilariae revealed they were neither D. immitis nor any other previously characterized North American species. PCR amplification of the 5S spacer region of rDNA revealed that the filarial sequence shared only 76% identity with D. immitis. This previously unidentified filarial sequence was present in all antigen positive animals (11 of 11 tested). It appears that black-footed ferrets were infected with a previously undescribed species of filaria whose antigen cross-reacted with the ELISA assay, although further analysis is needed to make a conclusive statement. Nonetheless, this previously undescribed filaria does not appear to threaten recovery for this highly endangered mammal.